APOLIPOPROTEIN ANALOGUES

Abstract

a pharmaceutical composition comprising an apolipoprotein construct, to an apolipoprotein construct, a nucleic acid sequence encoding the apolipoprotein construct, a vector comprising the nucleic acid sequence, a method for producing the apolipoprotein construct, and a method of treatment comprising administering the apolipoprotein construct. The presented data document that the constructs according to the invention are capable of binding lipids, are capable of binding cubilin, which is a strong Apo AI receptor, stronger than native Apo A-I and that the plasma half life of the constructs is at least tripled compared to native Apo A-I. Together these data document that the constructs according to the invention are strong candidates for treatment of cardiovascular diseases.

Description

[0001]The invention relates to a pharmaceutical composition comprising an apolipoprotein construct, to an apolipoprotein construct, a nucleic acid sequence encoding the apolipoprotein construct, a vector comprising the nucleic acid sequence, a method for producing the apolipoprotein construct, and a method of treatment comprising administering the apolipoprotein construct.

PRIOR ART

[0002]In the following, the term Apo A or apolipoprotein A will be used to designate any of the three apolipoproteins, Apolipoprotein A I, Apolipoprotein A II, or Apolipoprotein A IV.

[0003]Cardiovascular diseases caused by atherosclerosis in the vessels is the most frequent cause of death in the industrialised countries of the World. One of the pathogenic factors causing atherosclerosis is the deposition of cholesterol in the vessel walls leading to plaque formation and eventually to arterosclerosis and increased risk of infarction.

[0004]Apolipoprotein A-1 (apo-A-1) is the main component of plasma HDL (high density lipoprotein), which is negatively correlated to the presence of arterosclerosis. There is strong experimental evidence that this effect is caused by so-called reverse cholesterol transport from peripheral tissues to the liver. There is also experimental evidence that this reverse cholesterol transport can be stimulated in mammals by injection of apo-A-1.

[0005]Apolipoprotein A-1 is rapidly cleared from plasma. It is believed that Apo-A-1 is to a large extent removed from plasma by filtration in the kidneys without being broken down first (Braschi et al 1999, J Lipid Res, 40:522-532; Braschi et al 2000, Biochemistry, 39:5441-5449; Glass et al 1983, J Biol Chem 258:7161-7167). The short plasma half-life of apolipoprotein A is a constraint against using the protein in the treatment of atherosclerosis.

[0006]U.S. Pat. No. 5,876,968 (SIRTORI ET AL.) concerns substantially pure dimers of a variant of apo-A-1 called apolipoprotein A-1-Milano. Medicaments containing the dimer can be used for preventing thrombosis or they can be used as a prodrug for the monomer. A specific feature of this particular variant of apo-A-I is its ability to form covalent dimers with itself. The authors speculated that the presence of Apo A-I-M may be responsible for a prolonged plasma half-life, but no conclusive data have been presented.

[0007]U.S. Pat. No. 5,643,757 (SHA-IL ET AL.) discloses a method for the production of pure, stable, mature and biologically active human apolipoprotein A-I in high yield.

[0008]U.S. Pat. No. 5,990,081 (AGELAND ET AL.) discloses a method for treatment of arterosclerosis or cardiovascular diseases by administering a therapeutically effective amount of apoliproprotein A or apolipoprotein E.

[0009]WO 96/37608 (RHONE-POULENC ROHRER ET AL.) describes human homologous dimers of apolipoprotein A-I variants comprising cystein in position 151. The presence of the cystein residue in the amino acid sequence allows the formation of dimers via disulphide bridges between the monomers. The reference furthermore discloses the corresponding nucleic acid sequences and vectors comprising these as well as pharmaceutical compositions comprising the variants and the use of these in gene therapy.

[0010]WO 90/12879 (Sirtori et al) and WO 94/13819 (Kabi Pharmacia) disclose methods for preparation of ApoA-I and ApoA-IM in yeast and E. coli respectively. The documents also disclose the use of ApoA-I and ApoA-IM as a medicament for the treatment of atherosclerosis and cardiovascular diseases.

[0011]In conclusion the prior art is mainly concerned with the use of native ApoA-I or ApoA-IM monomer or ApoA-IM dimer as medicaments for the treatment of vascular diseases, despite the known disadvantages of these proteins (mainly rapid clearance). The prior art does not suggest to modify ApoA-I in order to obtain constructs with increased ability to perform reverse cholesterol transport and/or with longer plasma half life. It is thus one object of the present invention to provide such ApoA constructs, which may be used for treatment and/or prevention of cardiovascular diseases.

SUMMARY

[0012]In a first aspect the invention relates to a pharmaceutical composition comprising an apolipoprotein construct having the general formula [0013]apo A-X, [0014]where apo A is an apolipoprotein A component selected from the group consisting of apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, an analogue or a variant thereof, [0015]and X is a heterologous moiety comprising at least one compound selected from the group consisting of an amino acid, a peptide, a protein, a carbohydrate, and a nucleic acid sequence, [0016]with the proviso that when the construct consists of exactly two identical, native apolipoproteins these are linked serially.

[0017]By the invention is provided a novel pharmaceutical composition. The prior art fails to teach an apoliprotein construct as defined in the present invention for inclusion in a pharmaceutical composition. The apolipoprotein constructs according to the present invention may broadly be looked upon as HDL analogues due to their ability to form complexes with cholesterol and other lipids and assist in the transportation of these compounds to the liver.

[0018]Throughout the invention the apolipoprotein component or part of the construct is referred to as apo A or apolipoprotein. In the following and in the claims, the heterologous moiety is referred to as component X of the construct. The apolipoprotein or analogue or variant thereof is linked covalently to the heterologous moiety.

[0019]The component X of the construct may be looked broadly upon as a heterologous moiety. In this context a heterologous moiety is any kind of moiety not being linked to apolipoprotein or analogue or variant or functional equivalent thereof under native conditions. The heterologous moiety may thus be a peptide or a protein or part of a peptide or protein from the same or from another species, or even a single amino acid. It may be a synthetic peptide. It may be of carbohydrate nature or of other polymeric and biocompatible nature such as polyols, nucleic acids sequences.

[0020]Functional equivalence to native apolipoprotein A-I, A-II or A-IV may conveniently be measured using a lipid binding assay. The ability of the construct to elicit substantially the same physiological response in a mammal may conveniently be measured by measurement of the ability to perform reverse cholesterol transport in a test organism such as rabbits or rodent such as mice.

[0021]The construct comprising apolipoprotein and a heterologous moiety is capable of performing reverse cholesterol transport as well as or even better than native apolipoproteins, despite the modification caused by the addition of a heterologous moiety. The plasma half-life of the construct is preferably increased compared to that of the wild-type apolipoprotein. The increased half-life can be due either to the increased size of the apolipoprotein construct, which may reduce the rate of filtration through the kidneys, it may be due to increased binding to HDL, or it may be due to reduced breakdown of the construct compared to native Apo A.

[0022]Preferably the plasma half-life is at least doubled or tripled, or at least quadrupled, or at least 10 doubled. Similarly, the binding affinity such as the lipid binding affinity, and/or the cholesterol binding affinity of the construct is preferably increased as compared to wild-type apolipoprotein. Preferably, the lipid binding affinity is increased by at least 5%, such as at least 10%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 40% such as at least 50%, for example at least 75%, such as at least 100%, such as at least 150%, for example at least 200%, such as at least 300%. Even in the cases where the lipid binding affinity of the constructs according to the invention is the same or lower than the lipid binding affinity of native apolipoprotein, the clinical effect may be enhanced due to increased plasma half life of the constructs according to the invention.

[0023]An increased plasma half-time and/or increased lipid binding affinity have profound implications for the use of the apolipoprotein constructs in the treatment of arterosclerosis. It is therefore expected that the clinical effect of the apolipoprotein constructs according to the invention is superior to the effect of wild-type apolipoproteins.

[0024]The invention also encompasses analogues or variants of the wild-type apoliproteins capable of eliciting substantially the same physiological response in a mammal.

[0025]The pharmaceutical composition may further comprise pharmaceutical acceptable excipients, adjuvants, additives, such as lipids, phospholipids, cholesterol, or triglycerides.

[0026]According to a second aspect of the invention, there is provided an apolipoprotein construct having the general formula [0027]apo A-X, [0028]where apo A is an apolipoprotein component selected from the group consisting of apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, an analogue or a variant thereof, [0029]and X is a heterologous moiety selected from the group consisting of an oligomerising module, and a terminally linked apolipoprotein.

[0030]According to a further aspect, there is provided a nucleotide sequence encoding an apolipoprotein construct as defined above. Preferably the nucleotide sequence is operably linked to a regulatory sequence for expression of the protein construct.

[0031]According to further aspects of the invention, there is provided a vector comprising the nucleotide sequence encoding the apolipoprotein construct and a transformed host cell comprising the nucleotide sequence as defined above.

[0032]The apolipoprotein construct according to the invention may be produced by different methods.

[0033]According to a first method a transformed host cell is cultured under conditions promoting the expression of a protein construct according to the invention encoded by DNA inserted into a construct, obtaining and recovering the protein construct and optionally further processing the protein construct.

[0034]This method is the preferred method when the whole construct is of polypeptide nature and thus can be encoded by one corresponding nucleic acid sequence.

[0035]According to a second method the apolipoprotein construct can be manufactured by chemically synthesising the heterologous moiety and subsequently linking it to the apolipoprotein or analogue obtaining an apolipoprotein construct, which is isolated and optionally processed further. This method is the preferred method, when the heterologous moiety is of non-peptide nature. However there may also be conditions under which it is preferred to synthesise the heterologous moiety chemically, when it is of polypeptide nature. Such conditions may be that the heterologous moiety is rather short such as below 20 amino acids.

[0036]According to a third method the apolipoprotein construct can be manufactured by culturing a transformed host cell under conditions promoting the expression of an apolipoprotein or an apolipoprotein analogue encoded by a nucleic acid fragment and subsequently covalently linking the apolipoprotein or apolipoprotein analogue to a heterologous moiety obtaining an apolipoprotein construct, isolating the resulting apolipoprotein construct and optionally further processing the construct.

[0037]Finally, the apolipoprotein construct may be produced by culturing a transformed host cell under conditions promoting the expression of a protein encoded by a nucleic acid fragment encoding an oligomerising module and subsequently linking said module to at least one apolipoprotein obtaining an apolipoprotein construct.

[0038]According to a further aspect of the invention there is provided a method for treating a patient having a condition related to cholesterol, phospholipids and triacylglycerides LDL and HDL disorders, and arteriosclerotic diseases comprising administering to the individual a pharmaceutical composition according to the invention.

[0039]The pharmaceutical composition may be administered intravenously, intraarterially, intramusculary, transdermally, pulmonary, subcutaneously, intradermally, intratechally, through the buccal-, anal-, vaginal-, conjunctival-, or intranasal tissue, or by inoculation into tissue, such as tumour tissue, or by an implant, or orally.

[0040]The apolipoprotein construct as defined above may also be used for gene therapy, wherein the DNA sequence encoding the apolipoprotein construct is used for transfection or infection of at least one cell population.

DETAILED DESCRIPTION OF THE INVENTION

[0041]In the following the invention will be described in detail with reference to the following figures.

[0042]FIG. 1 shows the amino acid sequence (in one letter code) of human apolipoprotein A-I.

[0043]FIG. 2A shows CLUSTAL W (1.74) multiple sequence alignment of apolipoprotein A-I using BLOSUM. The following sequences are aligned in the Figure:

HUMAN sp|P02647|APA1_HUMAN Apolipoprotein A-I precursor (Apo-AI)--Homo sapiens (Human)Macaque sp|P15568|APA1_MACFA Apolipoprotein A-I precursor (Apo-AI)--Macaca fascicularis (Crab eating macaque)Bovine sp|P15497|APA1_BOVIN Apolipoprotein A-I precursor (Apo-AI)--Bos taurus (Bovine).Pig sp|P186481APA1_PIG Apolipoprotein A-I precursor (Apo-AI)--Sus scrofa (Pig).Dog sp|P02648|APA1_CANFA Apolipoprotein A-1 precursor (Apo-AI)--Canis familiaris (Dog).Rabbit sp|P09809|APA1_RABIT Apolipoprotein A-1 precursor (Apo-A1)--Oryctolagus cuniculus (Rabbit).Tree shrew sp|O18759|APA1TUPGB Apolipoprotein A-1 precursor (Apo-AI)--Tupaia glis belangeri (Common tree shrew).Mouse sp|Q00623|APA1_MOUSE Apolipoprotein A-1 precursor (Apo-AI)--Mus musculus (Mouse).Rat sp|P04639|APA1_RAT Apolipoprotein A-1 precursor (Apo-AI)--Rattus norvegicus (Rat).Eur. Hedgehog tr|Q9TS49 APOLIPOPROTEIN A-I, APOA-I═CHOLESTEROL TRANSPORTER--Erinaceus europaeus (Western European hedgehog).Chicken sp|P08250|APA1_CHICK Apolipoprotein A-I precursor (Apo-AI)--Gallus gallus (Chicken).Jap. quail sp|P32918|APA1_COTJA Apolipoprotein A-I precursor (Apo-AI)--Coturnix coturnix japonica (Japanese quail).Domestic duck sp|O42296|APA1_ANAPL Apolipoprotein A-I precursor (Apo-AI)--Anas platyrhynchos (Domestic duck).Rainbow trout sp|057523|AP11_ONCMY Apolipoprotein A-I-1 precursor (APOA-I-1) --Oncorhynchus mykiss (Rainbow trout) (Salmo gairdneri).Brown trout sp|Q91488|APA1_SALTR Apolipoprotein A-I precursor (ApoAI)--Salmo trutta (Brown trout).Atl. salmon sp|P27007|APA1_SALSA Apolipoprotein A-I precursor (Apo-AI)--Salmo salar (Atlantic salmon).Zebrafish sp|O42363|APA1_BRARE Apolipoprotein A-I precursor (Apo-AI)--Brachydanio rerio (Zebrafish) (Zebra danio).Sea bream sp|O42175|APA1_SPAAU Apolipoprotein A-I precursor (Apo-AI)--Sparus aurata (Gilthead sea bream).

[0044]FIG. 2B shows aligned amino acid sequences (in one letter code) for human, macaque, mouse, baboon, pig, and rat apolipoprotein A-IV.

[0045]FIG. 3: Amino acid sequence of the amino terminal region of tetranectin (SEQ ID NO 12). Amino acid sequence (in one letter code) from E1 to L51 of tetranectin. Exon 1 comprises residues E1 to D16 and exon 2 residues V17 to V49, respectively. The alpha helix extends beyond L51 to K₅₂ which is the C-terminal amino acid residue in the alpha helix.

[0046]FIG. 4 shows an alignment of the amino acid sequences of the trimerising structural element of the tetranectin protein family. Amino acid sequences (one letter code) corresponding to residue V17 to K₅₂ comprising exon 2 and the first three residues of exon 3 of human tetranectin; murine tetranectin (Sorensen et al., Gene, 152: 243-245, 1995); tetranectin homologous protein isolated from reefshark cartilage (Neame and Boynton, 1992, 1996); and tetranectin homologous protein isolated from bovine cartilage (Neame and Boynton, database accession number PATCHX:u22298). Residues at a and d positions in the heptad repeats are listed in boldface. The listed consensus sequence of the tetranectin protein family trimerising structural element comprise the residues present at a and d positions in the heptad repeats shown in the figure in addition to the other conserved residues of the region. "hy" denotes an aliphatic hydrophobic residue.

[0047]FIG. 5 shows the pT7H6UbiFx Apo A-I plasmid and its corresponding amino acid sequences. The expressed and processed polypeptide consists of amino acids no 25-267 from human Apo A-1 (SEQ ID NO 1) and gly-gly linked N-terminally thereto.

[0048]FIG. 6 shows the pT7H6UbiFx Cys-Apo A-1 plasmid and its corresponding amino acid sequences for. The expressed and processed polypeptide consists of a N-terminal cystein residue and the amino acids no 25-267 from human Apo A-1 (SEQ ID NO 2) and gly-gly linked N-terminally thereto.

[0049]FIG. 7 shows the pT7H6 Trip-A-Apo A-I-AmpR plasmid and its corresponding amino acid sequence. The expressed and processed polypeptide (SEQ ID NO 3) consists of the TTSE, a linking sequence, and amino acids no 25-267 from human Apo A-I.

[0050]FIG. 8 shows the pT7H6 Trip-A-Apo A-I-del 43-AmpR plasmid and its corresponding amino acid sequence. The expressed and processed polypeptide (SEQ ID NO 4) consists of the TTSE, a linking sequence, and amino acids no 68-267 from human Apo A-I.

[0051]FIG. 9 shows the pT7H₆FXCysApoAI plasmid and its corresponding amino acid sequence. The expressed and processed polypeptide consists of a N-terminal cystein residue and the amino acids no 25-267 from human Apo A-1 (SEQ ID NO 2) and gly-gly linked N-terminally thereto.

[0052]FIG. 10 A to G shows illustrative examples of plasmids and corresponding amino acid sequences for apolipoprotein constructs according to the present invention.

[0053]FIG. 10 A: pT7H6-Trip-A-Apo AI K₉A K15A: Corresponds to pT7H6-Trip-A-Apo AI but two lysine residues in the trimerisation region has been mutated to remove the heparin affinity. The mature protein product is called Trip-A-AI K₉A,K₁₅A (SEQ ID NO 5).

[0054]FIG. 10 B: pT7H6 Trip-A-FN-Apo AI: Corresponds to pT7H6-Trip-A-Apo AI, however, bases encoding the amino acid sequence SGH has been inserted after the Trip A sequence and before the apo AI sequence. The mature protein product is named Trip-A-FN-AI (SEQ ID NO 6).

[0055]FIG. 10 C: pT7H6 Trip-A-FN-Apo AI-final: Corresponds to pT7H6 Trip-A-FN-Apo AI, however, the BamHI site of pT7H6 Trip-A-FN-Apo AI has been removed and the inserted three amino acid sequence changed, so that the amino acid sequence between the tetranectin derived trimerisation sequence and apo AI has been changed from GSSGH to GTSGQ. The five amino acid sequence corresponds to a sequence in the linker region of fibronectin. The mature protein product is named Trip-A-FN-AI-finai (SEQ ID NO 7).

[0056]FIG. 10 D: pT7H6 Trip-A-FN-Apo AI-final K₉AK15A: Corresponds to pT7H6-Trip-A-FN-Apo AI-final but two lysine residues in the trimerisation region has been mutated to remove the heparin affinity. The mature protein product is called Trip-A-FN-AI-final-K₉A,K₁₅A (SEQ ID NO 8).

[0057]FIG. 10 E: pT7H6 Trip-A-TN-Apo AI: Corresponds to pT7H6-Trip-A-Apo AI, however, bases encoding the amino acid sequence KVHMK has been inserted after the Trip A sequence and before the apo AI sequence. The mature protein product is named Trip-A-TN-AI (SEQ ID NO 9).

[0058]FIG. 10 F: pT7H6 Trip-A-TN-Apo AI-final: Corresponds to pT7H6 Trip-A-TN-Apo AI, however, the BamHI site of pT7H6 Trip-A-TN-Apo AI has been removed so that the amino acid sequence between the tetranectin derived trimerisation sequence and apo AI has been changed from GSKVHMK to GTKVHMK. The seven amino acid sequence corresponds to the sequence of tetranectin following the trimerisation domain. The mature protein product is named Trip-A-TN-AI-final (SEQ ID NO 10).

[0059]FIG. 10 G: pT7H6 Trip-A-TN-Apo AI-final K₉AK15A: Corresponds to pT7H6-Trip-A-TN-Apo AI-final but two lysine residues in the trimerisation region has been mutated to remove the heparin affinity. The mature protein product is called Trip-A-TN-AI-final-K₉A,K₁₅A (SEQ ID NO 11).

[0060]FIG. 10 H: pT7H₆Fx-Hp(alpha)-ApoAI. The plasmid codes for the fusion protein between Hp(alpha) and ApoAi. The mature protein product is called Hp(alpha)-ApoAI (SEQ ID NO 14).

[0061]FIG. 11 shows the result of binding of ApoA-I, TripA-ApoA-I, and TripA-FN-ApoA-1 to DMPC in the assay described in Example 6.

[0062]FIG. 12 shows binding of ApoA-1 and TripA-ApoA-1 to immobilised cubilin as described in Example 7.

[0063]FIG. 13 shows analytical gelfiltration of Apo A-I, Trip-A-AI, Trip-A-TN-AI, Trip-A-FN-AI. As controls BSA was included. Details are disclosed in Example 5.

[0064]FIG. 14 shows the results of the evaluation of plasma clearance of apolipoprotein A-I, TripA Apo-AI, and TripA-fibronectin-linker Apo A-1 in mice. Experimental details can be found in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

[0065]The functionality of the constructs according to the invention and of the apo-A components of the constructs can be measured by a lipid binding assay such as by the DPMC assay described below. Furthermore, the in vivo effect on reverse cholesterol transport may be measured by administration to test animals such as rabbits fed on a cholesterol rich diet such as the method disclosed in Miyazaki et al (Arteriosclerosis, Thrombosis, and Vascular Biology, 1995; 15:1882-1888) or in Apo E deficient mice (Sha P K et al, Circulation 2001, 103:3047-3050).

The Apolipoprotein or Analogue

[0066]In the following the term "apo-A" is used to designate any apolipoprotein A comprising apolipoprotein A-I, apolipoprotein A-II or apolipoprotein A-IV, any variant or analogue thereof possessing the same lipid binding function. Preferred apolipoprotein A-1 analogues include those disclosed in FIG. 2A. Preferred apolipoprotein A-IV analogues include those disclosed in FIG. 2B. Known variants of the sequences of human Apo-AI in FIG. 1 include the following variants, indicating the position of the variation with respect to the sequence in FIG. 1, the variation, and where appropriate the name of the known variant.

TABLE-US-00001 27 P -> H (IN MUNSTER-3C). 27 P -> R. 28 P -> R (IN MUNSTER-3B). 34 R -> L (IN BALTIMORE). 50 G -> R (IN IOWA). 84 L -> R (IN AUTOSOMAL DOMINANT AMYLOIDOSIS). 113 D -> E. 119 A -> D (IN HITA). 127 D -> N (IN MUNSTER-3A). 131 MISSING (IN MARBURG/MUNSTER-2). 131 K -> M. 132 W -> R (IN TSUSHIMA). 133 E -> K (IN FUKUOKA). 151 R -> C (PARIS) 160 E -> K (IN NORWAY). 163 E -> G. 167 P -> R (IN GIESSEN). 168 L -> R (IN ZARAGOZA). 171 E -> V. 189 P -> R. 197 R -> C (IN MILANO). 222 E -> K (IN MUNSTER-4).

[0067]According to the invention the term "apolipoprotein" is meant to include functional equivalents of at least one sequence in FIGS. 1, 2a and 2b, or a fragment of at least one sequence in FIGS. 1, 2a and 2b, comprising a predetermined amino acid sequence. A "fragment" is defined as: [0068]i) fragments comprising an amino acid sequence capable of being recognised by an antibody also capable of recognising the predetermined amino acid sequences in FIGS. 1, 2a or 2b, and/or ii) fragments comprising an amino acid sequence capable of binding to a lipid such as dimyristoyl phosphatidylcholine or cholesterol, and/or a receptor, which is also capable of binding the predetermined amino acid sequences in FIGS. 1, 2a or 2b.

[0069]According to the present invention a functional equivalent of an apolipoprotein or fragments thereof may be obtained by addition, substitution or deletion of at least one amino acid. When the amino acid sequence comprises a substitution of one amino acid for another, such a substitution may be a conservative amino acid substitution. Fragments of the sequences in FIGS. 1, 2a and 2b may comprise more than one such substitution, such as e.g. two conservative amino acid substitutions, for example three or four conservative amino acid substitutions, such as five or six conservative amino acid substitutions, for example seven or eight conservative amino acid substitutions, such as from 10 to 15 conservative amino acid substitutions, for example from 15 to 25 conservative amino acid substitution, such as from 25 to 75 conservative amino acid substitutions, for example from 75 to 125 conservative amino acid substitutions, such as from 125 to 175 conservative amino acid substitutions. Substitutions can be made within any one or more groups of predetermined amino acids.

[0070]Examples of fragments comprising one or more conservative amino acid substitutions including one or more conservative amino acid substitutions within the same group of predetermined amino acids, or a plurality of conservative amino acid substitutions, wherein each conservative substitution is generated by substitution within a different group of predetermined amino acids.

[0071]Accordingly, a variant of the sequences in FIG. 1, 2a or 2b, or fragments thereof according to the invention may comprise, within the same variant of the sequences in FIG. 1, 2a or 2b, or fragments thereof or among different variant of the sequences in FIG. 1, 2a or 2b, or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently of one another. Variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof may thus comprise conservative substitutions independently of one another, wherein at least one glycine (Gly) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof of the sequences in FIG. 1, 2a or 2b is substituted with an amino acid selected from the group of amino acids consisting of Ala, Val, Leu, and Ile, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one of said alanines (Ala) of said variant of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Val, Leu, and Ile, and independently thereof, variant of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one valine (Val) of said variant of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Leu, and Ile, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one of said leucines (Leu) of said variant of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val, and Ile, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one isoleucine (Ile) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val and Leu, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof wherein at least one of said aspartic acids (Asp) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Glu, Asn, and Gln, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one of said phenylalanines (Phe) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Tyr. Trp, H is, Pro, and preferably selected from the group of amino acids consisting of Tyr and Trp, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one of said tyrosines (Tyr) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof of the sequences in FIG. 1, 2a or 2b is substituted with an amino acid selected from the group of amino acids consisting of Phe, Trp, H is, Pro, preferably an amino acid selected from the group of amino acids consisting of Phe and Trp, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one of said arginines (Arg) of said fragment of the sequences in FIG. 1, 2a or 2b is substituted with an amino acid selected from the group of amino acids consisting of Lys and H is, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one lysine (Lys) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Arg and H is, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one of said aspargines (Asn) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, Glu, and Gln, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one glutamine (Gln) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, Giu, and Asn, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one proline (Pro) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Phe, Tyr, Trp, and H is, and independently thereof, variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof, wherein at least one of said cysteines (Cys) of said variants of the sequences in FIG. 1, 2a or 2b, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, Glu, Lys, Arg, H is, Asn, Gln, Ser, Thr, and Tyr.

[0072]It is clear from the above outline that the same variant or fragment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.

[0073]The addition or deletion of an amino acid may be an addition or deletion of from 2 to 10 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as additions from 10 to 200 amino acids, are also comprised within the present invention. More specifically, 43 N-terminal amino acids may be removed from the sequence in FIG. 1 without substantially altering the lipid binding effect of the protein. Such a deletion variant is included in SEQ ID NO 4 as the apolipoprotein part of the construct.

[0074]It will thus be understood that the invention concerns apolipoproteins comprising at least one fragment of the sequences in FIG. 1, 2a or 2b capable of binding lipids such as DPMC, including any variants and functional equivalents of such at least one fragment.

[0075]The apolipoprotein according to the present invention, including any functional equivalents and fragments thereof, may in one embodiment comprise less than 243 amino acid residues, such as less than 240 amino acid residues, for example less than 225 amino acid residues, such as less than 200 amino acid residues, for example less than 180 amino acid residues, such as less than 160 amino acid residues, for example less than 150 amino acid residues, such as less than 140 amino acid residues, for example less than 130 amino acid residues, such as less than 120 amino acid residues, for example less than 110 amino acid residues, such as less than 100 amino acid residues, for example less than 90 amino acid residues, such as less than 85 amino acid residues, for example less than 80 amino acid residues, such as less than 75 amino acid residues, for example less than 70 amino acid residues, such as less than 65 amino acid residues, for example less than 60 amino acid residues, such as less than 55 amino acid residues, for example less than 50 amino acid residues.

Fragments

[0076]A fragment comprising the lipid binding region of the native sequences in FIG. 1, 2a or 2b is particularly preferred. However, the invention is not limited to fragments comprising the lipid binding region. Deletions of such fragments generating functionally equivalent fragments of the sequences in FIG. 1, 2a or 2b comprising less than the lipid binding region are also comprised in the present invention. Functionally equivalent the sequences in FIG. 1, 2a or 2b peptides, and fragments thereof according to the present invention, may comprise less or more amino acid residues than the lipid binding region. Preferably, the fragment comprises at least the amino acids 100-186 of apo-A-1 or a variant or a functional equivalent thereof. It has been determined that this central domain and the α-helices within the domain are directly involved in interactions with phospholipids. Therefore, it is highly likely that this region plays an important role in the functional properties of apo-A-I.

[0077]"Functional equivalency" as used in the present invention is according to one preferred embodiment established by means of reference to the corresponding functionality of a predetermined fragment of the sequences in FIG. 1, 2a or 2b. Functional equivalents of variants of the sequences in FIG. 1, 2a or 2b will be understood to exhibit amino acid sequences gradually differing from the preferred predetermined sequence, as the number and scope of insertions, deletions and substitutions including conservative substitutions increases. This difference is measured as a reduction in homology between the preferred predetermined sequence and the fragment or functional equivalent.

[0078]All fragments or functional equivalents of apolipoprotein are included within the scope of this invention, regardless of the degree of homology that they show to a preferred predetermined sequence of apolipoprotein. The reason for this is that some regions of the sequences in FIG. 1, 2a or 2b are most likely readily mutatable, or capable of being completely deleted, without any significant effect on the binding activity of the resulting fragment.

[0079]A functional variant obtained by substitution may well exhibit some form or degree of native activity of the sequences in FIG. 1, 2a or 2b, and yet be less homologous, if residues containing functionally similar amino acid side chains are substituted. Functionally similar in this respect refers to dominant characteristics of the side chains such as hydrophobic, basic, neutral or acidic, or the presence or absence of steric bulk. Accordingly, in one embodiment of the invention, the degree of identity between i) a given the sequences in FIG. 1, 2a or 2b fragment capable of effect and ii) a preferred predetermined fragment, is not a principal measure of the fragment as a variant or functional equivalent of a preferred predetermined the sequences in FIG. 1, 2a or 2b fragment according to the present invention. The homology between amino acid sequences may be calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90. Preferably the algorithm BLOSUM 30 is used.

[0080]Fragments sharing at least some homology with the sequences in FIG. 1, 2a or 2b fragment are to be considered as falling within the scope of the present invention when they are at least about 40 percent homologous with the apolipoprotein or fragment thereof, such as at least about 50 percent homologous, for example at least about 60 percent homologous, such as at least about 70 percent homologous, for example at least about 75 percent homologous, such as at least about 80 percent homologous, for example at least about 85 percent homologous, such as at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with the sequences in FIG. 1, 2a or 2b fragment. According to one embodiment of the invention the homology percentages refer to identity percentages.

[0081]Additional factors that may be taken into consideration when determining functional equivalence according to the meaning used herein are i) the ability of antisera against one of the sequences in FIG. 1, 2a or 2b to detect fragments of the sequences in FIG. 1, 2a or 2b according to the present invention, or ii) the ability of the functionally equivalent fragment to compete with the sequences in FIG. 1, 2a or 2b in a lipid binding assay.

[0082]Conservative substitutions may be introduced in any position of a preferred predetermined apolipoprotein or fragment thereof. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution in any one or more positions.

[0083]A non-conservative substitution leading to the formation of a functionally equivalent fragment of the sequences in FIG. 1, 2a or 2b would for example i) differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met) substituted for a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, H is or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as H is, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and vice versa).

[0084]Substitution of amino acids may in one embodiment be made based upon their hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, feucine and isoleucine.

[0085]In addition to the variants described herein, sterically similar variants may be formulated to mimic the key portions of the variant structure and that such compounds may also be used in the same manner as the variants of the invention. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.

The Component X

[0086]Preferably, the component X of the protein construct according to the invention is essentially non-immunogenic. For instance the component X may be an amino acid, a carbohydrate, a nucleic acid sequence, an inert protein or polypeptide, which has substantially no physiological effect and especially no immunological effect on mammals.

[0087]Preferably the component X is non immunogenic and does not interfere negatively with regard to ligand binding, i.e. the apolipoprotein component should not be directed at an undesired site through interactions of the X-component with a ligand. According to one embodiment the component X consists of just one amino acid, which amino acid preferably is a cystein residue, which may be placed N-terminally, C-terminally or internally in the apolipoprotein component. Such a construct may form a dimer with other identical or similar constructs. Preferably a linker is introduced between the terminal cystein residue and the apolipoprotein component to facilitate the correct folding and lipid interaction of the construct. However, more preferably the component X comprises a peptide having more than 1 amino acids such as more than 2 amino acids, for example more than 5 amino acids, such as more than 10 amino acids, for example more than 15 amino acids, such as more than 20 amino acids, such as more than 30 amino acids, for example more than 40 amino acids, such as more than 50 amino acids, for example more than 75 amino acids, such as more than 100 amino acids, for example more than 200 amino acids, such as more than 300 amino acids, for example more than 400 amino acids, such as more than 500 amino acids, for example more than 600 amino acids, such as more than 700 amino acids, for example more than 800 amino acids, such as more than 900 amino acids, for example more than 1000, 1250, 1500, 2000, or 2500 amino acids.

[0088]In the case where the X-component is a protein, this protein is preferably a mammalian protein and more preferably a human protein. Examples of suitable proteins include plasma proteins such as albumin or serum albumin or another non-immunogenic peptide or protein such as the serine protease fragment of plasminogen or another serine protease engineered to be inactive by disruption of the catalytic triad; and the constant region of the heavy chain of immunoglobins. More preferably, the protein comprises serum albumin. Even more preferably the protein comprises an apolipoprotein containing an amphipatic helix containing apolipoprotein.

[0089]According to an especially preferred embodiment of the invention, the component X comprises an apolipoprotein component selected from the group consisting of apolipoprotein A-I, A-II, AIV, an analogue, functional variant or fragment thereof. The two apolipoprotein components may be linked linearly or they may be linked via an additional non-native terminal cystein bridge.

[0090]Higher oligomers as well as dimers of the apolipoprotein component comprising at least one non-native cystein residue may be manufactured and linked through cystein bridges under appropriate conditions. Oligomers linked by disulphide bridges may be linked serially (apo-A-S--S-apo-A, or apo-A-S--S-apo-A-S--S-apo-A or higher oligomers).

[0091]The protein construct according to the invention may also comprise two, three or more apolipoproteins or analogues thereof being serially and covalently linked to one another. This may be achieved by linking the C-terminal of a first apolipoprotein to the N-terminus of the next apolipoprotein and so forth. The proteins may be so linked after transcription and translation or the nucleotide sequence may simply comprise two, three or more sequences coding for the apolipoprotein construct in question as well as optional linker peptides between the apolipoproteins. Thereby, the need for a heterologous moiety to perform the linkage is avoided. It is expected that in the constructs having two, three or more apo-A units essentially all the apo-A units will participate in lipid binding thereby contributing to the functionality of the construct. Therefore it is expected that these multi-apo-A constructs have an increased lipid binding ability compared to native apo-A. An additional advantage of these constructs compared to native apo-A, is that they have an increased plasma half-life compared to native apo-A.

[0092]Such constructs comprising more than one apolipoprotein component may comprise a combination selected from the following group:

TABLE-US-00002 Dimers: A-I A-I; A-II AII; A-IV A-IV; A-I A-II; A-I A-IV; A-II A-IV. Trimers: A-I A-II A-IV; A-I A-I A-II; A-I A-I A-I; A-I A-I A-IV; A-II A-II A-I; A-II A-II A-IV; A-II A-II A-II; A-IV A-IV A-IV; A-IV A-IV A-II; A-IV A-IV A-I.

Oligomerisation Modules

[0093]According to an especially preferred embodiment of the invention, the heterologous moiety is an oligomerising module. In this context, an oligomerising module is a peptide or a protein or part of a protein which is capable of interacting with other, similar or identical oligomerising modules. The interaction is of the type that produces multimeric proteins or polypeptides. Such an interaction may be caused by covalent bonds between the components of the multimer as well as by hydrogen bond forces, hydrophobic forces, van der Waals forces, salt bridges. The invention also encompasses oligomerising modules of non-peptide nature such as a nucleic acid sequence of DNA, RNA, LNA, or PNA. The skilled person is familiar with techniques to link proteins and nucleic acid sequences to one another. The oligomerisation module may be a dimerising module, a trimerising module, a tetramerising module, or a multimerising module.

[0094]When the apolipoprotein or analogue part of the construct is coupled to an oligomerising module, multimers of the construct can be made by simply mixing a solution of constructs (oligomerisation module linked to apolipoprotein part) under appropriate conditions. In this way, dimers, trimers, tretramers, pentamers, hexamers or higher -mers can be made depending on the type of oligomerising module being linked to the apolipoprotein part of the construct. The multimers according to the invention may be homomers or heteromers, since different apolipoproteins can be linked to the oligomerising modules and be incorporated into the multimer. It may be advantageous to mix the different types of apolipoproteins in this way to obtain an improved clinical effect of the construct. Preferred homomers include trimers of Apo-A-1 and trimers of Apo-A-IV. According to an especially preferred embodiment of the invention the oligomerising module is from tetranectin and more specifically comprises the tetranectin trimerising structural element (hereafter termed TTSE, SEQ ID NO 12), which is described in detail in WO 98/56906. The amino acid sequence of TTSE is set forth in SEQ ID NO 12. The trimerising effect of TTSE is caused by a coiled coil structure which interacts with the coiled coil structure of two other TTSEs to form a trimer, which is exceptionally stable. A further advantage of TTSE is that it is a weak antigen (WO 98/56906).

[0095]Preferably the heparin binding site, which is located in the N-terminal region of exon 1 (FIG. 4) is abolished by removal or mutagenis of N-terminal lysine residues (residues 9 and 14 of SEQ ID NO 12) (Nielsen et al, 1997, FEBS Lett 412:388-396) without inhibiting trimerisation. Preferably the lysine residues are mutagenised to alanine. TTSEs that include most or all of exon 1 therefore confer an affinity for sulfated polysaccharides to any designed protein which encompasses such a TTSE as part of its structure. If desired, however, this affinity can be reduced or abolished by N-terminal truncation or mutagenesis of lysine residues in the part of the TTSE that corresponds to the N-terminal amino acid residues of tetranectin (Lorentsen et al 2000, Biochem J 347:83-87).

[0096]The interacting domain of the trimerising module according to the invention is preferably of the same type as in TTSE, namely a triple alpha helical coiled coil. The TTSE may be from human tetranectin, from rabbit tetranectin, from murine tetranectin or from C-type lectin of shark cartilage Preferably, the TTSE comprises a sequence having at least 68%, such as at least 75%, for example at least 81%, for example at least 87% such as at least 92% identity with the consensus sequence of SEQ ID NO 12. Thereby analogues of the TTSE having substantially the same trimerising effect are encompassed by the invention.

[0097]Preferably, the cystein residue 50 of TTSE (SEQ ID NO 12) should be mutagenised to serine, threonine, methionine or to any other amino acid residue in order to avoid formation of an unwanted inter-chain disulphide bridge, which could lead to unwanted multimerisation.

[0098]The presence of a trimer may be ascertained by well known techniques such as gel-filtration, SDS-PAGE, or native SDS gel electrophoresis depending on the nature of the trimer. One preferred method for ascertaining the presence of an oligomer is through linkage by DMSI (dimethylsubirimidate) followed by SDS-PAGE. According to a preferred embodiment of the invention the protein construct is obtained by linking two or more apolipoproteins to oligomerising modules. The advantage of this embodiment is that the linkage of the individual apolipoproteins to one another does not take place within the apolipoprotein but in the oligomerising module. Thereby the nature of the wild-type apolipoprotein is conserved and the apolipoprotein conserves the secondary and tertiary structure, which is advantageous for its physiological function. By further introducing a peptide spacer between the apolipoprotein and the oligomerising module it is ensured that both of the components of the construct can perform their interaction with lipids and other oligomerising modules respectively without being affected by the interactions of the other component. Preferably, the peptide spacer is non-immunogenic, and has an essentially linear three dimensional structure.

[0099]Different or identical apo-A units may be oligomerised using an oligornerisation module such as a dimerising module, a trimerising module, a tetramerising module, a pentamerising module, a hexamerising module or a multimerising module. The oligomerising modules may comprise a coiled coil structure capable of interchain recognition and interaction.

[0100]The general method for producing an artificial trimer of a protein or peptide comprises the identification of a trimerisation module from proteins that form trimers in nature. Through careful analysis, the domain responsible for the protein-protein interaction can be identified, isolated, and linked to the protein or peptide to be trimerised. According to the invention such trimerisation does not necessarily comprise the formation of a trimer of apolipoprotein or an analogue. It is also possible to link just one apolipoprotein to a trimerisation module and allow this peptide to trimerise with two other trimerisation modules. Thereby the molecular weight of the apolipoprotein part is increased and the plama half-life may be increased compared to native apolipoprotein.

[0101]One example of an oligomerisation module is disclosed in WO 95/31540 (HOPPE ET AL.), which describes polypeptides comprising a collectin neck region. The amino acid sequence constituting the collectin neck region may be attached to any polypeptide of choice. Trimers can then be made under appropriate conditions between three polypeptides comprising the collectin neck region amino acid sequence.

[0102]Another example of an oligomerisation module is the (1-chain from Haptoglobin. The α1-chain has a cystein residue which may link to another α1-chain to form a dimer. A natural variant is the α2-chain, which has had part of the (1-chain involved in disulphide bridging duplicated. The α2-chain may form cystein bridges to cystein residues in other α2 or α1-chains thereby forming trimers, tetramers, pentamers, hexamers and higher -mers. In the natural form the α-chain is associated to a β-chain. It is possible to replace the β-chain with an apolipoprotein to make an apo-A-α-chain (haptoglobin) construct.

Spacer Peptide

[0103]The protein construct may also advantageously comprise a spacer moiety, which is covalently linked between the apolipoprotein or apolipoprotein analogue and the heterologous moiety. The effect of the spacer is to provide space between the heterologous moiety and the apolipoprotein part of the construct. Thereby is ensured that the secondary structure of the apolipoprotein part is not affected by the presence of the heterologous moiety so that the physiological effect of the apolipoprotein part is maintained. Preferably, the spacer is of polypeptide nature. In this way the nucleic acid sequence encoding the spacer can be linked to the sequence encoding the apolipoprotein part of the construct and optionally the sequence for the heterologous moiety, and the whole construct can be produced at the same time.

[0104]Design and preparation of suitable spacer moieties are known in the art and are conveniently effected by preparing fusion polypeptides having the format apo-A-spacer-X, where the spacer moiety is a polypeptide fragment (often a relatively inert one), so as to avoid undesired reactions between the spacer and the surroundings or the construct.

[0105]A spacer moiety may also be inserted between two TTSEs allowing both of these to interact with a third separate TTSE to form a trimeric complex, which then comprises two separate peptides: TTSE and TTSE-spacer-TTSE. This embodiment facilitates the production of the apolipoprotein construct since the major part of the trimer, which is then strictly seen a dimer, can be synthesised as one single polypeptide comprising in the fusion partners (apo-A denoting any polypeptide sequence forming the apolipoprotein part of the construct) apo-A-TTSE-spacer-TTSE-apo-A. In the embodiments where two TTSEs are present in the same monomer it is preferred that the spacer moiety has a length and a conformation which favours complex formation involving both of the two TTSEs which are covalently linked by the spacer moiety. In this way, problems arising from undesired formation of trirners of the formats (2+1+1), (2+2+2), and (2+2+1) (wherein only one TTSE of each monomer participates in complex formation) can be diminished.

[0106]The spacer peptide preferably comprises at least two amino acids, such as at least three amino acids, for example at least five amino acids, such as at least ten amino acids, for example at least 15 amino acids, such as at least 20 amino acids, for example at least 30 amino acids, such as at least 40 amino acids, for example at least 50 amino acids, such as at least 60 amino acids, for example at least 70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids such as approximately 100 amino acids.

[0107]The spacer may be linked to the apo-A component and X through covalent linkages. and preferably the spacer is essentially non-immunogenic, and/or is not prone to proteolytic cleavage, and/or does not comprise any cystein residues. Similarly, the three-dimensional structure of the spacer is preferably linear or substantially linear.

[0108]The following are examples of spacer sequences, which are believed to be especially preferable for linking apolipoprotein analogues to a component X. Preferred examples of spacer or linker peptides include those, which have been used to link proteins without substantially impairing the function of the linked proteins or at least without substantially impairing the function of one of the linked proteins. More preferably the linkers or spacers have been used to link proteins comprising coiled-coil structures.

Tetranectin Based Linker:

[0109]The linker may include the tetranectin residues 53-56, which in tetranectin forms a β-strand, and the residues 57-59 which forms a turn in tetranectin (Nielsen B B, Kastrup J S, Rasmussen H, Holtet T L, Graversen J H, Etzerodt M, Thogersen H C, Larsen I K, FEBS-Letter 412, 388-396, 1997). The sequence of the segment is GTKVHMK. This linker has the advantage that it in native tetranectin is bridging the trimerisation domain with the CRD-domain, and hence is imagined to be well suited for connecting the trimerisation domain to another domain in general. Furthermore the resulting construct is not expected to be more immunogenic than the construct without a linker. The tetranectin based linker is highly preferred when the component X comprises the TTSE.

Fibronectin Based Linker:

[0110]The linker may be chosen as a sub-sequence from the connecting strand 3 from human fibronectin, this corresponds to amino acid residues 1992-2102 (SWISS--PROT numbering, entry PO₂₇₅₁). Preferably the subsequence: PGTSGQQPSVGQQ covering amino acid residues number 2037-2049 is used, and within that subsequence the segment GTSGQ corresponding to amino acid residues 2038-2042 is more preferable. This construct has the advantage that it is know not to be highly prone to proteolytic cleavage and is not expected to be highly immunogenic bearing in mind that fibronectin is present at high concentrations in plasma.

Human IgG₃ Upper Hinge Based Linker

[0111]The 10 amino acid residue sequence derived from the upper hinge region of murine IgG₃, PKPSTPPGSS, has been used for the production of antibodies dimerised trough a coiled coil (Pack P. and Pluckthun, A. Biochemistry 31, pp 1579-1584 (1992)) and may be useful as a spacer peptide according to the present invention. Even more preferable may be a corresponding sequence from the upper hinge region of human IgG₃. Sequences from human IgG₃ are not expected to be immunogenic in human beings.

Flexible Linkers

[0112]Possible examples of flexible linker/spacer sequences include SGGTSGSTSGTGST, AGSSTGSSTGPGSTT or GGSGGAP. These sequences have been used for the linking of designed coiled coils to other protein domains (Muller, K. M., Arndt, K. M. and Alber, T., Meth. Enzymology, 328, pp 261-281 (2000).

The Linkage

[0113]The two components of the construct may be linked together by a covalent linkage. This linkage may be formed between the component X and the C or N terminal amino acid of the apo-A component. The components may also be linked via more than one covalent linkages. The covalent linkage between the components may also comprise a S--S bridge, preferably between cystein residues. These cystein residues is placed C or N terminally in the apo-A component and terminally or internally in the component X.

Carbohydrate

[0114]Irrespective of the other components of the construct the construct according to the invention may comprise a carbohydrate moiety.

Tetranectin trimerising structural element One especially preferred embodiment of the invention is the trimerisation or partial trimerisation of an apolipoprotein or analogue thereof with the trimerisation module from tetranectin.

[0115]This technique is described in WO 98/56906 (THOGERSEN ET AL.), which is hereby incorporated by reference. The trimeric polypeptides are constructed as a monomer polypeptide construct comprising at least one tetranectin trimerising structural element (TTSE), which is covalently linked to at least one heterologous moiety. The tetranectin trimerising structural element is capable of forming a stable complex with two other tetranectin trimerising structural elements. The term "trimerising structural element" (TTSE) used in the present description and claims is intended to refer to the portion of a polypeptide molecule of the tetranectin family which is responsible for trimerisation between monomers of the tetranectin polypeptide (SEQ ID NO 12). The term is also intended to embrace variants of a TTSE of a naturally occurring tetranectin family member, variants which have been modified in the amino acid sequence without adversely affecting, to any substantial degree, the trimerisation properties relative to those of the native tetranectin family member molecule.

[0116]Specific examples of such variants will be described in detail herein, but it is generally preferred that the TTSE is derived from human tetranectin, murine tetranectin, C-type lectin of human or bovine cartilage, or C-type lectin of shark cartilage. Especially preferred is monomer polypeptide constructs including at least one TTSE derived from human tetranectin.

[0117]The 51 residue polypeptide sequence encoded by exons 1 and 2 of tetranectin (FIG. 3, SEQ ID NO 12) appears to be unique to the tetranectin group of proteins (FIG. 4) as no significant sequence homology to other known polypeptide sequences has been established. In preparation for experimental investigations of the architecture of tetranectin a collection of recombinant proteins have been produced, the collection including complete tetranectin, the CRD domain (approximately corresponding to the polypeptide encoded by exon 3), a product corresponding to the polypeptide encoded by exons 2+3, a product corresponding to exons 1+2 (Holtet et al., 1996). Tetranectin is indeed a trimer, but the exon 2 encoded polypeptide is in fact capable of effecting trimerisation by itself as evidenced by the observation that the recombinant protein corresponding to exons 2+3 is in fact trimeric in solution.

[0118]3D-structure analysis of crystals of full-length recombinant tetranectin (Nielsen et al., 1996; Nielsen, 1996; Larsen et al., 1996; Kastrup, 1996) has shown that the polypeptide encoded in exon 2 plus three residues encoded in exon 3 form a triple alpha helical coiled coil structure.

[0119]From the combination of sequence and structure data it becomes clear that trimerisation in tetranectin is in fact generated by a structural element (FIG. 4), comprising the amino acid residues encoded by exon two and the first three residues of exon 3 by an unusual heptad repeat sequence, that apparently is unique to tetranectin and other members of its group: This amino acid sequence (FIG. 4) is characterised by two copies of heptad repeats (abcdefg) with hydrophobic residues at a and d positions as are other alpha helical coiled coils. These two heptad repeats are in sequence followed by an unusual third copy of the heptad repeat, where glutamine 44 and glutamine 47 not only substitute the hydrophobic residues at both the a and d position, but are directly involved in the formation of the triple alpha helical coiled coil structure. These heptad repeats are additionally flanked by two half-repeats with hydrophobic residues at the d and a position, respectively. The presence of beta-branched hydrophobic residues at a or d positions in alpha helical coiled coil are known to influence the state of oligomerisation. In the tetranectin structural element only one conserved valine (number 37) is present. At sequence position 29 in tetranectin no particular aliphatic residue appears to be preferred.

[0120]In summary, it is apparent that the triple stranded coiled coil structure in tetranectin to a large extent is governed by interactions that are unexpected in relation to those characteristic among the group of known coiled coil proteins.

[0121]The TTSEs form surprisingly stable trimeric molecules. The experimental observations, that (1) a substantial part of the recombinant proteins exists in the oligomeric state of and can be cross-linked as trimeric molecules even at 70° C.° and (2) that exchange of monomers between different trimers can only be detected after exposure to elevated temperature are evidence of a extremely high stability of the tetranectin trimerising structural element. This feature must be reflected in the amino acid sequence of the structural element. In particular, the presence and position of the glutamine containing repeat in the sequential array of heptad repeats is, together with the presence and relative position of the other conserved residues in the consensus sequence (FIG. 4), considered important for the formation of these stable trimeric molecules. For most practical uses the cysteine residue 50 should be mutagenized to serine, threonine, methionine or to any other amino acid residue in order to avoid formation of an unwanted inter-chain disulphide bridge, which may lead to uncontrolled multimerisation, aggregation and precipitation of a polypeptide product harbouring this sequence.

[0122]In particular in conjunction with the trimer-stabilising exon 1 encoded polypeptide, the tetranectin trimerising structural element is a truly autonomous polypeptide module retaining its structural integrity and propensity to generate a highly stable homotrimeric complex whether it is attached or not by a peptide bond at either or at both termini to other proteins.

[0123]This unique property is demonstrated by the fact that polypeptide sequences derived from heterologous proteins may readily be trimerised when joined as fusion proteins to the tetranectin trimerising structural element. This remains valid irrespective of whether the heterologous polypeptide sequences are placed amino-terminally or carboxy-terminally to the trimerising element allowing for the formation of one molecular assembly containing up to six copies of one particular polypeptide sequence or functional entities, or the formation of one molecular assembly containing up to six different polypeptide sequences, each contributing their individual functional property.

[0124]Since three TTSEs of naturally occurring human tetranectin forms up a triple alpha helical coiled coil, it is preferred that the stable complex formed by the TTSEs of the invention also forms a triple alpha helical coiled coil. 32 The "tetranectin family" are polypeptides, which share the consensus sequence shown in FIG. 4 or a sequence, which is homologous at sequence level with this consensus sequence.

[0125]Hence, monomer polypeptide constructs of the invention are preferred which comprise a polypeptide sequence which has at least 68% sequence identity with the consensus sequence shown in FIG. 4, but higher sequence identities are preferred, such as at least 75%, at least 81%, at least 87%, and at least 92%.

Trip A-Module

[0126]In the expression plasmids according to the present invention, the TTSE module (SEQ ID NO 12) was modified as indicated by replacing Cys 50 by Ser and including a C-terminal lysin residue. A SPGT sequence has been added to the N-terminal. This is a connective sequence to the trimerisation module. The sequence has been inserted because it gives the opportunity to cut the DNA strand with Bgl II and Kpn K. C-terminally a connective GS sequence has been added, which provides an opportunity to cut with Bam HI. This modified TTSE is designated TripA and disclosed as SEQ ID NO 13. The trimerisation module of the Apo A construct may thus advantageously comprise this sequence or a sequence haveng at least 68% sequence identity with the sequence of SEQ ID NO 13, but higher sequence identities are preferred, such as at least 75%, at least 81%, at least 87%, and at least 92%.

[0127]Specific examples of constructs encompassing the Trip A module are disclosed in the examples.

EXAMPLES OF CONSTRUCTS ACCORDING TO THE INVENTION

[0128]The invention encompasses the specific sequences disclosed in the appended examples as SEQ ID NO 2 to 11 and SEQ ID NO 14. Preferably the invention encompasses SEQ ID NO 3 to 11 and SEQ ID NO 14. Sequences sharing at least 60% sequence identity, such as at least 70% sequence identity to these sequences are also within the scope of the invention, preferably sequences sharing at least 80% sequence identity, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

Production of the Protein Construct

[0129]In order to produce a peptide component of the protein construct the cDNA encoding this part is inserted into an expression vector and transformed into a host cell.

[0130]The above mentioned host cell (which is also a part of the invention) can be prepared by traditional genetic engineering techniques which comprises inserting a nucleic acid fragment (normally a DNA fragment) encoding the polypeptide part of a monomer polypeptide construct of the invention into a suitable expression vector, transforming a suitable host cell with the vector, and culturing the host cell under conditions allowing expression of the polypeptide part of the monomer polypeptide construct. The nucleic acid fragment encoding the polypeptide may be placed under the control of a suitable promoter which may be inducible or a constitutive promoter. Depending on the expression system, the polypeptide may be recovered from the extracellular phase, the periplasm or from the cytoplasm of the host cell. Suitable vector systems and host cells are well-known in the art as evidenced by the vast amount of literature and materials available to the skilled person. Since the present invention also relates to the use of the nucleic acid fragments of the invention in the construction of vectors and in host cells, the following provides a general discussion relating to such use and the particular considerations in practising this aspect of the invention.

[0131]In general, of course, prokaryotes are preferred for the initial cloning of nucleic sequences of the invention and constructing the vectors useful in the invention. For example, in addition to the particular strains mentioned in the more specific disclosure below, one may mention by way of example, strains such as E. coli K₁₂ strain 294 (ATCC No. 31446), E. coli B, and E. coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative rather than limiting. Prokaryotes are also preferred for expression, since efficient purification and protein refolding strategies are available. The aforementioned strains, as well as E. coli W3110 (F-λ, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may be used.

[0132]In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977). The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.

[0133]The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.

[0134]Those promoters most commonly used in recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979; EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilised, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.

[0135]In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiase, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980).

[0136]This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0137]Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

[0138]Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilisation. Any plasmid vector containing a yeast compatible promoter, origin of replication and termination sequences is suitable.

[0139]In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure (Tissue Culture, 1973). Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293 and MDCK cell lines.

[0140]Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

[0141]For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BgII site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilise promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

[0142]An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

[0143]Upon production of the polypeptide monomer constructs it may be necessary to process the polypeptides further, e.g. by introducing non-proteinaceous functions in the polypeptide, by subjecting the material to suitable refolding conditions (e.g. by using the generally applicable strategies suggested in WO 94/18227), or by cleaving off undesired peptide moieties of the monomer (e.g. expression enhancing peptide fragments which are undesired in the end product).

[0144]In the light of the above discussion, the methods for recombinantly producing the monomer polypeptide construct of the invention are also a part of the invention, as are the vectors carrying and/or being capable of replicating the nucleic acids according to the invention in a host cell or a cell-line. According to the invention the expression vector can be e.g. a plasmid, a cosmid, a minichromosome, or a phage. Especially interesting are vectors which are integrated in the host cell/cell line genome after introduction in the host.

[0145]Another part of the invention are transformed cells (useful in the above-described methods) carrying and capable of replicating the nucleic acid fragments of the invention; the host cell can be a microorganism such as a bacterium, a yeast, or a protozoan, or a cell derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. Especially interesting are cells from the bacterial species Escherichia, Bacillus and Salmonella, and a preferred bacterium is E. coli.

[0146]Yet another part of the invention relates to a stable cell line producing the polypeptide part of a construct according to the invention, and preferably the cell line carries and expresses a nucleic acid of the invention.

Receptor Binding

[0147]The performance of the constructs according to the invention may be analysed by measuring the ability of the constructs to bind to receptors or HDL proteins which may bind native apolipoprotein A-I, A-II or A-IV. Such receptors and proteins include but are not limited to cubilin, megalin, Scavenger receptor class B type 1 (SR-B1), ATP-binding cassette 1 (ABC1), Lecithin:cholesterol acyltransferase (LCAT), Cholesteryl-ester transfer protein (CETP), Phospolipid transfer protein (PLTP). The dissociation constant, Kd, of the complex between cubilin and native apolipoprotein A I is 20 nM. It has been determined experimentally that an apolipoprotein A I trimer according to the present invention binds even stronger to cubilin (FIG. 12).

Affinity Tags

[0148]The protein construct according to the invention may also comprise an affinity tag for use during purification of the construct. Such a tag preferably comprises a polyhistidine sequence. This sequence can advantageously be used for purification of the product on a Ni²+ column, which will bind the polyhistidine sequence and thereby the whole protein. After elution from the column the polyhistidine sequence may be cleaved off by a proteinase such as trombin recognising a specific sequence built into the construct between the protein construct and the polyhistidine sequence.

[0149]Other examples of affinity tags include but are not limited to well known tags such as an antigenic tag, or a GST tag. A proteolytic cleavage site may be inserted between the tag and the construct to cleave off the tag.

Signal Peptides

[0150]When expressing the constructs according to the invention in E. coli or in yeast, it may be preferable to include a signal peptide in the expression construct to ensure that the expressed protein is secernated and can be harvested from the medium surrounding the cells instead of the more laborious process of isolating the expressed protein from within the cells. Specific examples of signal peptides for expression in yeast and E. coli, which can be used in conjunction with the present invention include those disclosed in WO 90/12879 (Sirtori et al), which discloses a signal peptide for expression of Apo-AI and Apo-AIM in yeast, and WO 94/13819 (Kabi Pharmacia) disclosing a signal peptide for expression of Apo-AI and Apo-AIM in E. coli.

Production of apo-A-TTSE

[0151]In order to produce a construct comprising an apolipoprotein part and a TTSE, the cDNA encoding the apolipoprotein part is ligated at the 3' end to the 5' end of the c-DNA encoding the TTSE. Further TTSE units and apolipoprotein units may also be ligated. A sequence encoding an enzyme cleavage site is further ligated to the 3' end of the sequence encoding TTSE and finally a sequence encoding polyhistidine is also ligated. This can be done by conventional PCR techniques. The combined c-DNA is inserted into an expression vector and transformed into a host cell.

[0152]After expression in the E. coli, the polyhistidine sequence is used to capture the heterologous protein on a Ni2+ column. After elution the polyhistidine tail can be removed by a proteinase such as Fx cleaving the heterologous protein at the specific site inserted into it between the TTSE and the polyhistidine sequence. The resulting apo-A-TTSE peptide can then be processed further by trimerising it to other or identical apo-A-TTSE peptides. To improve expression in E. coli it may be advantageous to express the construct as a fusion protein together with e.g. ubiquitin, which may be cleaved off later.

Use of an apo-A Construct for Preparation of a Pharmaceutical Composition

[0153]The apo-A construct may be used for the preparation of a pharmaceutical composition. The composition may comprise pharmaceutical acceptable excipients, adjuvants, additives such as phospholipids, cholesterol, or triglycerides.

[0154]The pharmaceutical composition may be administered intravenously, intraarterially, intramusculary, transdermally, pulmonary, subcutaneously, intradermally, intratechally, through the buccal-, anal-, vaginal-, conjunctival-, or intranasal tissue, or by inoculation into tissue, such as tumour tissue, or by an implant, or orally.

[0155]The formulation of the pharmaceutical compositions according to the invention is preferably performed using techniques well known to the skilled practitioner. This may comprise the addition of pharmaceutically acceptable excipients, adjuvants, or additives, such as phospholipids, cholesterol or triglycerides.

Administration of apo-A Construct

[0156]The apo-A-constructs according to the invention may be administered for prevention and/or treatment of diseases related to cholesterol, phospholipids, and triacylglycerides, LDL and HDL disorders such as hypercholesterolemia, and arteriosclerotic diseases such as atherosclerosis and myocardial infarct. Other indications include angina pectoris, plaque angina pectoris, unstable angina pectoris, arterial stenoses such as carotis stenosis, claudicatio, or cerebral arterial stenosis. Furthermore, the apolipoprotein constructs may be used for removal of endotoxins.

[0157]In one embodiment, administration comprises the administration of at least 50 mg of the construct every week such as to obtain a plasma concentration of approximately 0.5 g/L. Preferably the construct is administered parenterally such as through injections, suppositories, implants etc. Preferably the composition is administered in an amount comprising at least 50 mg apolipoprotein construct per week, such as at least 100 mg/week, for example at least 250 mg/week, such as at least 500 mg/week, for example at least 750 mg/week such as at least 1000 mg/week, for example at least 1250 mg/week, such as at least 1500 mg/week, for example at least 2000 mg/week, such as at least 2500 mg/week, for example at least 5000 mg/week. The administration may be performed daily, every two or three days, once a week, once every second week, or once every third week, or once every fourth week.

[0158]According to another embodiment, the construct is administered once, twice or three times in much higher amounts especially for acute treatment of angina pectoris and plaque angina pectoris or unstable angina pectoris. The administration may be performed during 1, 2, 3, 4, 5, 6, 7, 8 or up to 10 days. These amounts may be at least 10 mg/kg body weight, such as at least 20 mg/kg body weight, for example at least 30 mg/kg, such as at least 40 mg/kg, for example at least 50 mg/kg, such as at least 60 mg/kg, for example at least 70 mg/kg, such as at least 75 mg/kg, for example at least 90 mg/kg, such as at least 100 mg/kg, for example at least 125 mg/kg, such as at least 150 mg/kg, for example at least 200 mg/kg, such as at least 250 mg/kg, for example at least 300 mg/kg, such as at least 400 mg/kg, for example at least 500 mg/kg, such as at least 600 mg/kg, for example at least 700 mg/kg, such as at least 800 mg/kg, for example at least 900 mg/kg, such as at least 1000 mg/kg.

[0159]The constructs may also be administered orally. For this administration route, the technology described in WO 99/46283, U.S. Pat. No. 5,922,680, U.S. Pat. No. 5,780,434 or U.S. Pat. No. 5,591,433, U.S. Pat. No. 5,609,871, or U.S. Pat. No. 5,783,193 may be applied to the protein constructs according to the present invention. These references are hereby incorporated in their entirety by reference.

Cell Population

[0160]The invention also encompasses the use of the nucleotide sequence according to the invention for gene therapy.

[0161]The genes may be transferred to a population of macrophages and subsequently be transferred to the patient in need of treatment. Hereby, a transient expression of the gene is obtained, since the macrophage have a limited lifetime in the blood vessels.

[0162]Permanent transfection may be obtained by transforming liver cells.

[0163]The invention is now described with specific examples of embodiments of the invention, which are to be interpreted as illustrative rather than limiting examples. The design of further constructs according to the invention Ile within the normal skills of the practitioners within the art.

EXAMPLE 1

Cloning of Apo A-1

[0164]The cDNA encoding Apo A-I was amplified from a human liver cDNA library (Clontech) using standard PCR techniques. For the construction of Ubi-A-I the primers used were: 5'-CAC GGA TCC ATC GAG GGT AGG GGT GGA GAT GAA CCC CCC CAG AGC-3' and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3'. The product was cloned into the vector pT7H6Ubi, described in (Ellgaard L. et al Eur. J. Biochem. 1997; 244(2):544-51) using the Bam HI and Hind III cloning sites. For the construction of Trip-A-A-I the primers used were 5'-AAG GGA TCC GAT GAA CCC CCC CAG AGC CCC-3' and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3'. The PCR product was cloned into the pT7H6tripa vector described in WO 98/56906 using the Bam HI and Hind III cloning sites. For the construction of Trip-A-I-del43 the primers used were 5'-AGG GGA TOC CTA AAG CTC CTT GAC AAC TGG G-3' and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3'. The PCR product was cloned into the pT7H6tripa vector described in WO 98/56906 using the Bam HI and Hind III cloning sites. For the construction of Ubi-Cys-A-I the primers used were: 5'-GGT GGA TCC ATC GAG GGT AGG GOT GGA TGT GAT GAA CCC CCC C-3' and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3'. The product was cloned into the vector pT7H6Ubi, described in (Ellgaard L. et al Eur. J. Biochem. 1997; 244(2):544-51) using the Bam HI and Hind III cloning sites. The plasmids generated are shown on FIGS. 4, 5, 6, and 7.

EXAMPLE 2

Expression of Apolipoprotein A-I (apo A-I) in E. coli

[0165]Ubi-A-1 and Trip-A-I as well as the other constructs disclosed in the figures are conveniently expressed in E. coli AV-1 cells (Stratagene Inc.). Other cell lines may be used as well. Culturing of the cells and induction of expression were performed as described for tetranectin in WO 98/56906.

EXAMPLE 3

Isolation and Processing of Protein

[0166]Crude protein was isolated by phenol extraction as described for tetranectin in WO 98/56906. The re-dissolved pellet from 6 litres of expression culture was centrifuged to remove non-dissolved material and then batch adsorbed to 50 ml Ni²+--NTA-Sepharose, prepared as described in WO 98/56906. The column material was packed on a column and then washed with 500 ml 8 M urea, 500 mM NaCl, 50 mM Tris-HCl pH 8.0, then 200 ml of 6 M Guanidinium-HCl, 50 mM Tris-HCl pH 8.0 and finally 300 ml of 500 mM NaCl, 50 mM Tris-HCl pH 8.0. The protein was eluted with 500 mM NaCl, 50 mM Tris-HCl pH 8.0 and 10 mM EDTA. The protein was added 0.5 mg of Factor Xa and digested overnight at room temperature. Thrombin may be used for this purpose as well. The protein was gelfiltrated on a G-25 sephadex (Pharmacia) column in to a 500 mM NaCl, 50 mM Tris-HCl pH 8.0 buffer. Undigested protein was removed by passing the protein solution over a Ni²+--NTA-Sepharose column pre-washed in 500 mM NaCl, 50 mM Tris-HCl pH 8.0 and then washed with 500 mM NaCl, 50 mM Tris-HCl pH 8.0. Undigested protein was eluted with 500 mM NaCl, 50 mM Tris-HCl pH 8.0 and 10 mM EDTA. Further purification may be performed using Sp Sepharose ion exchange.

EXAMPLE 4

Removal of Lipids from the Proteins

[0167]The proteins were gelfiltrated into a 10 mM (NH₄)₂CO₃ pH 8.8 solution and lyophilised. The lyophilised protein was resuspended in 25 ml cold 1:1 methanol/chloroform, incubated on ice for 30 min, centrifuged at 3000 g for 20 minutes. The pellet was resuspended in 25 ml of 1:2 cold methanol/chloroform, equilibrated for 30 minutes on ice and recentrifuged. The supernatant was removed and the pellet was briefly air-dried and then redissolved in 6 M guanidinium-HCl, 50 mM Tris-HCl pH 8.0 over night.

EXAMPLE 5

Multimerisation Assay

Cross Linking

[0168]Multimerisation may be measured by cross-linking of multimers followed by analytical SDS-PAGE.

[0169]60 μl of a 0.2 mg/ml protein dissolved in 150 mM Na-borate pH 9.0 equillibrated to the desired temperature for 30 minutes are added 5 μl of a 20 mg/ml dimethylsuberimidate and incubated for 30 minutes at the desired temperature. The cross-linking was quenched by the addition of 5 μl 3 M Tris-HCl pH 9.0.

[0170]Dimethylsuberimidate causes lysin residues located within a short distance from one another to form a covalent bond. The result is that proteins which have formed multimers are covalently linked to one another. The molecular weight of the multimers can be estimated in the subsequent SDS-PAGE.

[0171]The cross-linking products were analysed by SDS-PAGE on 8-16% polyacrylamide gels. Optionally an adjuvant, such as a lipid, was included in the cross-linking mixture, in which case the protein was pre-incubated with the adjuvant.

Analytical Gelfiltration

[0172]Multimerisation may also be measured by analytical gelfiltration.

[0173]The protein was dissolved in a 500 mM NaCl, 50 mM Tris-HCl pH 8.0 buffer and gelfiltrated on a Superdex 200 HR 10/30 column in to the desired buffer at room temperature and a flow of 0.25 ml/min. For standard procedures the buffer was 100 mM NaCl, 50 mM Tris-HCl pH 8.0.

[0174]From FIG. 13 it can be seen that Apo A-I elutes as composite peaks, the major ones centred at approximately 14.5 and 16.5 mL. BSA, with a molecular weight of 68 kDa, elutes at approximately 14.5 ml, indicating that the Apo A-I peak at 16.5 ml corresponds to monomeric Apo A-I, while the other major peak corresponds to apo A-I self-association complexes. The constructs fused to the trimerisation domain all elute with a main peak at approximately 10.7 ml and a minor peak at 14 ml. The peak at 14 ml probably corresponds to the trimeric form of the constructs, while the main peak at 10.7 ml corresponds to a high molecular weight product. Presumably the product is formed by association of the trimers. This indicates that fusion of apo A-I to the trimerisation domain does not only lead to trimers, but also to the formation of large complexes, where the Apo A-I units can interact with other apo A-I units, like native apo A-I can interact with other Apo A-I molecules.

EXAMPLE 6

Kinetics of Association of the Protein Construct with Dimyristoyl Phosphatidylcholine (DMPC)

[0175]The ability of the constructs according to the invention to bind to a lipid can conveniently be measured using a well known assay such as the association to dimyristoyl phosphatidylcholine (DPMC).

[0176]The assay was conducted as described in (Bergeron J. et al. (1997), Biochem. Biophys. Acta, 1344, 139-152. Dried DPMC was suspended in 100 mM NaCl, 50 mM Tris-HCl pH 8.0 and 0.25 mM EDTA above its transition temparature at a concentration of 0.5 mg/ml. The protein sample, buffer and the DMPC suspension were all incubated at 24° C. 10 minutes, and then mixed so that the final concentration of DMPC became 0.4 mg/ml, with a protein conc. of 5.2 μM (of the monomer). The reduction in turbidity of the mixture, reflecting increasing lipid-protein association, was followed by measuring the absorbance of the mixture at 325 nm. The assay was conducted four times each for apo AI, Trip-A-AI and Trip-A-FN-AI, and one time without adding protein.

[0177]From FIG. 11 it can be seen that all Apo A-I constructs bind DMPC. For all the three constructs tested the turbidity was totally cleared after 24 hours, indicating that the capacity of the fusion proteins to bind DMPC is present in the fusion proteins. However, apparently both Trip-A-Apo A-I and Trip-A-FN-Apo AI binds DMPC slower than does native Apo A-I at 24° C., which is the only temperature at which the assay is functional.

EXAMPLE 7

Surface Plasmon Resonance Analysis of the Binding of the Derivatives to Cubilin

[0178]The assay was conducted as described in: Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen C, Cui S et al. The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein. Nat Med 1999; 5(6):656-661. The concentration of apolipoprotein construct used was 0.5 μM. Results (FIG. 12) are only shown for TripA-AI and apo A-I. Binding similar to that observed for TripA-AI was observed for TripA-FN-AI and TripA-TN-AI. The response increased upon trimerisation of apo A-I, especially there was a decrease in the of-rate, based on the gained "avidity" of the interaction for a multimer with an immobilised target compared to a monomer (bonus of multivalency). Showing that apo A-I was able to bind cubilin in the trimeric state, and that more than one apo A-I was in a conformation capable of interacting with cubilin, indicating correct folding of the apo A-I unit in the trimeric construct.

EXAMPLE 8

Evaluation of the Plasma Clearance of Apolipoprotein A-I, TripA Apo-A-1 and TripA Fibronectin-Linker Apo-A-1 in Mice

[0179]Three groups of five mice each were each injected 1 mg of apo A-I, Trip-A-AI or Trip-A-FN-AI, respectively. The protein was dissolved at a concentration of 0.33 mg/ml in the following buffer: 1×PBS pH 7.4, and 8.9 mg/ml dipalmitoylphosphatidylcholine. Blood samples were taken from each mice at the following times after the injection: 10 min., 4 h, 24 h, and 48 h.

[0180]The plasma concentrations of apolipoprotein A-I and derivatives were measured using an ELISA assay as follows:

[0181]Nunc Immuno PolySorp plates were used, each vial was added 100 μL in each addition. MB corresponds to the following buffer composition: 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 140 mM NaCl.

[0182]Plates were coated overnight in cold-room with 4 μg/ml of polygonal anti-human apo A-I from rabbit (DAKO A/S) dissolved in 50 mMNaHCO₃ pH 9.6. Plates were washed in MB+0.05% Tween-20 pH 7.8 and blocked in MB+0.05% Tween-20+1% BSA pH 7.8 for 1 hour. The sample was applied dissolved in MB+0.05% Tween-20+1% BSA pH 7.8 and incubated for 1 hour, washed in MB+0.05% Tween-20+1% BSA pH 7.8 and incubated for 1 hour with monoclonal anti-apo A-I from mice (Perlmmune Inc, clone 10-A8) at a concentration of 1 μg/ml in MB+0.05% Tween-20+1% BSA pH 7.8. Plates were washed and incubated with a secondary anti-mice IgG antibody linked to horse radish peroxidase. Plates were washed again and developed using OPD-tablets and H₂O₂, the reaction was stopped using 1 M H₂SO₄. The result was compared to a standard based on known concentrations of apo A-I and derivatives, respectively. No effect of diluting Apo A-I in mice plasma was observed for the standard.

[0183]The results, shown in FIG. 14, verify that the plasma clearance time of the construct Trip A Apo A-I is increased at least 3 times compared to the clearance time of native Apo AI. Preliminary data indicate that the clearance time for Trip A FN Apo A-I is at least the same as for Trip A Apo A-I. These data together with the cubilin binding data and DMPC binding data document that the constructs according to the invention are strong candidates for treating the diseases mentioned in the present application.

EXAMPLE 9

Plasmids

[0184]The construct according to the invention may be manufactured using the plasmids disclosed below.

Insertion of a Linker Sequence in Trip-A-AI

[0185]The basic linker containing constructs, with the mutations mentioned, was constructed as was the construct with-out linker. I.e. by PCR amplification of Apo A-I (and the linker sequence) and insertion into the pT7FxH6-Trip-A plasmid. The reverse primer was the same as used for the construction of pT7H₆FxTrip-A-AI, while the forward primers used were:

TABLE-US-00003 pT7H6FX-Trip-A-FN(-2)-AI: 5'-CGC GGATCC TCG GGT CAG GAT GAA CCC CCC CAG AGC CCC -3'

[0186]Unfortunately all the isolated clones had the above highlighted G mutated to a T, indicating a faulty sequence of the primer.

TABLE-US-00004 pT7H6FX-Trip-A-TN-AI-Bam-S 5'- cgc gga tcc aag gtg cac atg aag gat gaa ccc ccc cag agc ccc-3'

[0187]The mutations mentioned was corrected by site directed mutagenesis using the QuickChange kit from Stratagene and the following sets of primers:

TABLE-US-00005 pT7H6FXTrip-FN-AI: 5'-acg gtc tcc ctg aag gga acc tcg ggt cag gat g-3' 5'-cat cct gac ccg agg ttc cct tca ggg aga ccg t-3' pT7H6FX-Trip-A-TN-AI 5'-acg gtc tcc ctg aag gga acc aag gtg cac atg aag g-3' 5'-cct tca tgt gca cct tgg ttc cct tca ggg aga ccg t-3'

Removal of the Heparin-Binding Site of Trip-A

[0188]As a further derivation of the constructs, the heparin binding site of the Trip-A sequence (Lorentsen R H, Graversen J H, et al. Biochemical Journal (2000), 347 pp 83-87, was mutated using the site directed mutagenseis kit from Stratagene and the following set of primers:

TABLE-US-00006 For the mutation of lysine 9 from Trip-A: 5'-cca acc cag aag ccc aag gcg aat gta aat gcc-3' 5'-gtg ttc aca aca tct gcc ttg gca ttt aca atc-3' For the mutation of lysine 15 from Trip-A: 5'-ggc att tac aat cgc ctt ggg ctt ctg ggt tgg-3' 5'-cca acc cag aag ccc aag gcg att gta aat gcc-3'

[0189]These mutations are planned to be made on all the relevant Trip-A-apo-AI derivatives, possibly them all. Generating double mutants K9A, K15A of the trip-A derivatives, named TripA-FN-A1-K9AK15A, TripA-TN-AI-K9AK15A and TripA-AI-K9AK15A.

[0190]Furthermore truncation of the N-terminal could also remove the heparin affinity without removing the trimerisation. See Holtet et al. Protein Science (1997), Lorentsen et al. Biochem. Journal (2000), Nielsen et al. FEBS (1997) and Nielsen et al Acta Cryst D. (2000). The N-terminal residue would then preferably be located between Val 16 and Met 22.

Construction of the Expression Plasmid for Hp-α-AI

[0191]First the plasmid pT7H6Fx-Hp(alpha) (FIG. 10H) was constructed as follows:

[0192]From a cDNA library (Clontech fetal liver) the Hp-alpha sequence was PCR amplified using the following set of primers:

TABLE-US-00007 Non-sense primer: 5'-cac aag ctt tcc gct aga tct ctg cac tgg gtt agc cgg att ctt ggg -3' Sense Primer: 5'-ggt gga tcc atc gag ggt agg ggt gtg gac tca ggc aat gat gtc acg g-3' -3'

[0193]The PCR product contained the flowing features:

[0194]BamH I-site-Hp alpha sequence-BgI II site-Hind III site.

[0195]In the Hp-alpha sequence the cystein disulfide bridging with the beta-chain of Hp was mutated to an alanine.

[0196]The product was digested with BamH I and Hind III and inserted into the pT7H6FX plasmid, which was sequenced. A PCR product encoding human Apo AI was made using the standard non-sense primer also used for making pT7H6-Ubi-Fx-ApoAI and pT7H6Fx-TripA-AI. White the sense primer used was the one used as sense primer in the construction of pT7H6FX-TripA-AI. Giving a PCR product with the following features:

[0197]BamH I site-Apo AI sequence-Hind III site, this product was digested with BamH I and Hind III and inserted into the above mentioned plasmid digested with BgI II and Hind III. The resulting plasmid pT7H6FX-Hp(alpha)-Apo AI was sequenced and expression tested in E. coli.

pT7H6 TripA-apoAI (FIG. 7):

[0198]The plasmid comprises the plasmid pT7H6FxtripA described in WO 98/56906 as example no. 1.

[0199]Expression is governed by the T7 promoter. The plasmid furthermore comprises a H6 sequence being a hexa-His affinity tag for use in purification. After that is inserted a Factor Xa recognition sequence (IQGR). [0200]SPGT is a connective sequence to the subsequent trimerisation module. This sequence has been inserted because it gives the opportunity to cut the DNA strand with BgI II and Kpn I. [0201]Trip A is the trimerisation module from tetranectin.

[0202]GS is another connective sequence, which provides an opportunity to cut with Bam. HI.

[0203]Finally the plasmid comprises the human apoliprotein A-I cDNA coding for amino acids 25-267 from human apolipoprotein A-I. The expressed and purified protein corresponds to SEQ ID NO 3.

pT7H6TripA-apoAI-del43 (FIG. 8):

[0204]The plasmid comprises the sequences as above, but the apolipoprotein part has been replaced with cDNA coding for amino acids 68-267 from human apolipoprotein A-I. The expressed and purified protein corresponds to SEQ ID NO 4.

pT7H6UbiFxApoAI (FIG. 5):

[0205]The basic plasmid has been described in Ellgaard et al (1997).

[0206]The plasmid comprises the following sequences: [0207]the expression is governed by the T7 promoter [0208]H6: hexa-His affinity tag for purification of the protein construct [0209]Ubi: cDNA coding for human ubiquitin inserted to stabilise the protein in E. coli. [0210]FX: recognition sequence for Factor Xa [0211]DNA coding for two Gly residues, necessary for the optimal cleavage by Factor Xa. [0212]ApoAI: cDNA coding for amino acids 25-267 from human apolipoprotein A-I

[0213]The expressed and purified protein corresponds to SEQ ID NO 1.

pT7H6UbiFXCysApoAI (FIG. 6):

[0214]As above, but after the sequence coding for the two glycine residues and before the apolipoprotein A-I sequence coding for a cystein residue has been inserted. The expressed and purified protein corresponds to SEQ ID NO 2.

PT7H6FXCysApoAI (FIG. 9):

[0215]The plasmid comprises the following sequences: [0216]the expression is governed by the T7 promoter [0217]H6: hexa-His affinity tag for purification of the protein construct [0218]FX: recognition sequence for Factor Xa [0219]DNA coding for two Gly residues, necessary for the optimal cleavage by Factor Xa. [0220]DNA coding for a cystein residue. [0221]ApoAI: cDNA coding for amino acids 25-267 from human apolipoprotein A-I

[0222]The expressed and purified protein corresponds to SEQ ID NO 2.

[0223]Further examples of plasmids for expression of apolipoprotein constructs according to the invention are disclosed in FIG. 10 A to G together with the corresponding amino acid sequences of the expressed and purified proteins, which are disclosed in the sequence listing.

REFERENCES

[0224]Bergeron et al 1997, Biochem Biophys Acta, 1344:139-152. [0225]Bolivar et al, 1977. Gene, 2:95. [0226]Chang et al. 1978. Nature, 275: 617-624. [0227]Eligaard et al (1997). Dissection of the domain architecture of the α₂macroglobulin-receptor-associated protein. Eur J Biochem vol 244:544-551. [0228]Fiers et al. 1978. Nature, 273:113. [0229]Goeddel et al. 1979. Nature, 281:544. [0230]Hess et al. 1969. Advances in Enzyme Regulation, 7: 149-166. [0231]Hitzman et al. 1980. Journal of Biological Chemistry, 25: 12073-12080. [0232]Holland et al. 1978. Biochemistry, 17:4900. [0233]Holtet, T. L., Graversen, J. H., Thogersen, H. C. and Etzerodt, M. (1996).Domains and shared motifs in plasminogen-ligand interaction. Poster 21st Annual Lorne Conference on Protein Structure and Function, held Melbourne, Australia, Feb. 4-8, 1996. [0234]Itakura et al. 1977. Science, 198:1056. [0235]Jones. 1977. Genetics, 85: 23-33. [0236]Kastrup, J. S. (1996). Lecture at Minisymposium held by EU HCM contract CHRX-CT93-0143: Protein Crystallography I in Hamburg, Germany, Dec. 13-14, 1996. [0237]Kingsman et al, 1979, Gene.141. [0238]Larsen, I. K., Nielsen, B. B., Rasmussen, H. and Kastrup, J. S. (1996). Poster, 17th International Crystallography Congress, Seattle, USA held Aug. 8-17. 1996. [0239]Neame, P. J. and Boynton, R. E. (1996). Protein Soc. Symposium, (Meeting date 1995; 9th Meeting: Tech. Prot. Chem. VII). Proceedings pp. 401-407 (Ed., Marshak, D. R.; Publisher: Academic, San Diego, Calif.). [0240]Nielsen, B. B. (1996). Lecture, Lundbeck Centre Neuro-Medicinal Chemistry Minisymposium held Nov. 5, 1996 at the Royal Danish School of Pharmacy, Copenhagen. [0241]Nielsen, B. B., Larsen, I. K., Rasmussen, H. and Kastrup, J. S. (1996). Lecture, Danish Crystallographer's Meeting, held Jun. 3-4, 1996 at the Royal Danish School of Pharmacy, Copenhagen. [0242]Siebwenlist et al. 1980. Cell, 20:269. [0243]Sorensen et al, 1995, Gene, 152:243-245. [0244]Stinchomb et al. 1979. Nature 282:39. [0245]Tschemper et al. 1980. Gene, 10:157.

Sequence CWU 1

911243PRTHomo sapiens 1Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr1 5 10 15Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln20 25 30Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp35 40 45Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu50 55 60Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu65 70 75 80Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys85 90 95Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met100 105 110Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu115 120 125Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu130 135 140Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg145 150 155 160Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala165 170 175Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr180 185 190His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys195 200 205Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser210 215 220Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu225 230 235 240Asn Thr Gln2244PRTHomo sapiensmisc_feature(1)..(1)N-terminal Cys 2Cys Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala1 5 10 15Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser20 25 30Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu35 40 45Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln50 55 60Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr65 70 75 80Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala85 90 95Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu100 105 110Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln115 120 125Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro130 135 140Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu145 150 155 160Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala165 170 175Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu180 185 190Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala195 200 205Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu210 215 220Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys225 230 235 240Leu Asn Thr Gln3301PRTHomo sapiensmisc_feature(1)..(58)Trimerisation module from tetranectin 3Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn1 5 10 15Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Ser Asp Glu Pro Pro Gln Ser50 55 60Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu65 70 75 80Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu85 90 95Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr100 105 110Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu115 120 125Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met130 135 140Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp145 150 155 160Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys165 170 175Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu180 185 190His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp195 200 205Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr210 215 220Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys225 230 235 240Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu245 250 255His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu260 265 270Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu275 280 285Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln290 295 3004258PRTHomo sapiensmisc_feature(1)..(58)Trimerisation module from tetranectin 4Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn1 5 10 15Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Ser Leu Lys Leu Leu Asp Asn50 55 60Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly65 70 75 80Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly85 90 95Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val100 105 110Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu115 120 125Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly130 135 140Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly145 150 155 160Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr165 170 175His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg180 185 190Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His195 200 205Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro210 215 220Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe225 230 235 240Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn245 250 255Thr Gln5301PRTHomo sapiensmisc_feature(1)..(58)Trimerisation module from tetranectin 5Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala Ile Val Asn1 5 10 15Ala Lys Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Ser Asp Glu Pro Pro Gln Ser50 55 60Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu65 70 75 80Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu85 90 95Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr100 105 110Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu115 120 125Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met130 135 140Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp145 150 155 160Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys165 170 175Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu180 185 190His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp195 200 205Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr210 215 220Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys225 230 235 240Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu245 250 255His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu260 265 270Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu275 280 285Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln290 295 3006304PRTHomo sapiensmisc_feature(1)..(58)Trimerisation module from tetranectin 6Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn1 5 10 15Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Ser Ser Gly His Asp Glu Pro50 55 60Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val65 70 75 80Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly85 90 95Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp100 105 110Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val115 120 125Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg130 135 140Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro145 150 155 160Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr165 170 175Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg180 185 190Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu195 200 205Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu210 215 220Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu225 230 235 240Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys245 250 255Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu260 265 270Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val275 280 285Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln290 295 3007304PRTHomo sapiensmisc_feature(1)..(56)Trimerisation module from tetranectin 7Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn1 5 10 15Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Ser Gly Gln Asp Glu Pro50 55 60Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val65 70 75 80Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly85 90 95Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp100 105 110Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val115 120 125Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg130 135 140Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro145 150 155 160Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr165 170 175Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg180 185 190Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu195 200 205Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu210 215 220Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu225 230 235 240Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys245 250 255Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu260 265 270Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val275 280 285Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln290 295 3008304PRTHomo sapiensmisc_feature(1)..(56)Trimerisation module from tetranectin 8Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala Ile Val Asn1 5 10 15Ala Lys Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Ser Gly Gln Asp Glu Pro50 55 60Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val65 70 75 80Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly85 90 95Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp100 105 110Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val115 120 125Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg130 135 140Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro145 150 155 160Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr165 170 175Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg180 185 190Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu195 200 205Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu210 215 220Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu225 230 235 240Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys245 250 255Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu260 265 270Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val275 280 285Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln290 295 3009306PRTHomo sapiensmisc_feature(1)..(58)Trimerisation module from tetranectin 9Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn1 5 10 15Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Ser Lys Val His Met Lys Asp50 55 60Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val65 70 75 80Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe85 90 95Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn100 105 110Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly115 120 125Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly130 135 140Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val145 150 155 160Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu165 170 175Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly180 185 190Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly195 200 205Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr210 215 220His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg225 230 235 240Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His245 250 255Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro260 265 270Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe275 280 285Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn290

295 300Thr Gln30510306PRTHomo sapiensmisc_feature(1)..(56)Trimerisation module from tetranectin 10Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn1 5 10 15Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Lys Val His Met Lys Asp50 55 60Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val65 70 75 80Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe85 90 95Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn100 105 110Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly115 120 125Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly130 135 140Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val145 150 155 160Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu165 170 175Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly180 185 190Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly195 200 205Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr210 215 220His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg225 230 235 240Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His245 250 255Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro260 265 270Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe275 280 285Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn290 295 300Thr Gln30511306PRTHomo sapiensmisc_feature(1)..(56)Trimerisation module from tetranectin 11Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala Ile Val Asn1 5 10 15Ala Lys Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Lys Val His Met Lys Asp50 55 60Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val65 70 75 80Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe85 90 95Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn100 105 110Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly115 120 125Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly130 135 140Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val145 150 155 160Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu165 170 175Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly180 185 190Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly195 200 205Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr210 215 220His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg225 230 235 240Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His245 250 255Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro260 265 270Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe275 280 285Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn290 295 300Thr Gln3051251PRTHomo sapiens 12Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys Lys Asp1 5 10 15Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu Asp Thr20 25 30Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu Gln Thr35 40 45Val Cys Leu501358PRTHomo sapiensmisc_feature(1)..(4)Linker sequence 13Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn1 5 10 15Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser20 25 30Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln35 40 45Ala Leu Gln Thr Val Ser Leu Lys Gly Ser50 5514329PRTHomo sapiensmisc_feature(1)..(86)Hp(alpha) residues 14Gly Val Asp Ser Gly Asn Asp Val Thr Asp Ile Ala Asp Asp Gly Cys1 5 10 15Pro Lys Pro Pro Glu Ile Ala His Gly Tyr Val Glu His Ser Val Arg20 25 30Tyr Gln Cys Lys Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly Val35 40 45Tyr Thr Leu Asn Asn Glu Lys Gln Trp Ile Asn Lys Ala Val Gly Asp50 55 60Lys Leu Pro Glu Cys Glu Ala Val Ala Gly Lys Pro Lys Asn Pro Ala65 70 75 80Asn Pro Val Gln Arg Ser Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg85 90 95Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly100 105 110Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu115 120 125Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser130 135 140Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn145 150 155 160Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu165 170 175Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys180 185 190Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu195 200 205Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln210 215 220Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala225 230 235 240His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu245 250 255Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly260 265 270Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr275 280 285Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu290 295 300Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu305 310 315 320Glu Tyr Thr Lys Lys Leu Asn Thr Gln32515267PRTHomo sapiens 15Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser1 5 10 15Gln Ala Arg His Phe Trp Gln Gln Asp Glu Pro Pro Gln Ser Pro Trp20 25 30Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp35 40 45Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys50 55 60Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr65 70 75 80Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp85 90 95Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys100 105 110Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe115 120 125Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu130 135 140Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu145 150 155 160Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala165 170 175Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp180 185 190Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn195 200 205Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu210 215 220Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln225 230 235 240Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala245 250 255Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln260 26516267PRTMacaca fascicularis 16Met Lys Ala Thr Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser1 5 10 15Gln Ala Arg His Phe Trp Gln Gln Asp Glu Pro Pro Gln Thr Pro Trp20 25 30Asp Arg Val Lys Asp Leu Val Thr Val Tyr Val Glu Ala Leu Lys Asp35 40 45Ser Gly Lys Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys50 55 60Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr65 70 75 80Val Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp85 90 95Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys100 105 110Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe115 120 125Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu130 135 140Pro Leu Arg Ala Glu Leu His Glu Gly Thr Arg Gln Lys Leu His Glu145 150 155 160Leu His Glu Lys Leu Ser Pro Leu Gly Glu Glu Val Arg Asp Arg Ala165 170 175Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp180 185 190Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn195 200 205Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Ser Glu His Leu210 215 220Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln225 230 235 240Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala245 250 255Leu Glu Glu Tyr Thr Lys Lys Leu Ser Thr Gln260 26517265PRTBos Taurus 17Met Lys Ala Val Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser1 5 10 15Gln Ala Arg His Phe Trp Gln Gln Asp Asp Pro Gln Ser Ser Trp Asp20 25 30Arg Val Lys Asp Phe Ala Thr Val Tyr Val Glu Ala Ile Lys Asp Ser35 40 45Gly Arg Asp Tyr Val Ala Gln Phe Glu Ala Ser Ala Leu Gly Lys Gln50 55 60Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Thr Leu Ala Ser Thr Leu65 70 75 80Ser Lys Val Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp85 90 95Asn Leu Glu Lys Glu Thr Ala Ser Leu Arg Gln Glu Met His Lys Asp100 105 110Leu Glu Glu Val Lys Gln Lys Val Gln Pro Tyr Leu Asp Glu Phe Gln115 120 125Lys Lys Trp His Glu Glu Val Glu Ile Tyr Arg Gln Lys Val Ala Pro130 135 140Leu Gly Glu Glu Phe Arg Glu Gly Ala Arg Gln Lys Val Gln Glu Leu145 150 155 160Gln Asp Lys Leu Ser Pro Leu Ala Gln Glu Leu Arg Asp Arg Ala Arg165 170 175Ala His Val Glu Thr Leu Arg Gln Gln Leu Ala Pro Tyr Ser Asp Asp180 185 190Leu Arg Gln Arg Leu Thr Ala Arg Leu Glu Ala Leu Lys Glu Gly Gly195 200 205Gly Ser Leu Ala Glu Tyr His Ala Lys Ala Ser Glu Gln Leu Lys Ala210 215 220Leu Gly Glu Lys Ala Lys Pro Val Leu Glu Asp Leu Arg Gln Gly Leu225 230 235 240Leu Pro Val Leu Glu Ser Leu Lys Val Ser Ile Leu Ala Ala Ile Asp245 250 255Glu Ala Ser Lys Lys Leu Asn Ala Gln260 26518265PRTSus scrofa 18Met Lys Ala Val Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser1 5 10 15Gln Ala Arg His Phe Trp Gln Gln Asp Asp Pro Gln Ser Pro Trp Asp20 25 30Arg Val Lys Asp Phe Ala Thr Val Tyr Val Asp Ala Ile Lys Asp Ser35 40 45Gly Arg Asp Tyr Val Ala Gln Phe Glu Ala Ser Ala Leu Gly Lys His50 55 60Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Leu Gly Ser Thr Phe65 70 75 80Thr Lys Val Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp85 90 95Asn Leu Glu Lys Glu Thr Glu Ala Leu Arg Gln Glu Met Ser Lys Asp100 105 110Leu Glu Glu Val Lys Lys Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln115 120 125Asn Lys Trp Gln Glu Glu Met Glu Thr Tyr Arg Gln Lys Met Ala Pro130 135 140Leu Gly Ala Glu Phe Arg Glu Gly Ala Arg Gln Lys Val Gln Glu Leu145 150 155 160Gln Glu Lys Leu Ser Pro Leu Ala Glu Glu Leu Arg Asp Arg Leu Arg165 170 175Ala His Val Glu Ala Leu Arg Gln His Val Ala Pro Tyr Ser Asp Asp180 185 190Leu Arg Gln Arg Met Ala Ala Arg Phe Glu Ala Leu Lys Glu Gly Gly195 200 205Gly Ser Leu Ala Glu Tyr Gln Ala Lys Ala Gln Glu Gln Leu Lys Ala210 215 220Leu Gly Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu225 230 235 240Leu Pro Val Leu Glu Asn Leu Lys Val Ser Ile Leu Ala Ala Ile Asp245 250 255Glu Ala Ser Lys Lys Leu Asn Ala Gln260 26519266PRTCanis familiaris 19Met Lys Ala Ala Leu Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser1 5 10 15Gln Ala Arg His Phe Trp Gln Gln Asp Glu Pro Gln Ser Pro Trp Asp20 25 30Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Ala Val Lys Asp Ser35 40 45Gly Arg Asp Tyr Val Ala Gln Phe Glu Ala Ser Ala Leu Gly Lys Gln50 55 60Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Leu Ser Ser Thr Val65 70 75 80Thr Lys Leu Arg Glu Gln Ile Gly Pro Val Thr Gln Glu Phe Trp Asp85 90 95Asn Leu Glu Lys Glu Thr Glu Val Leu Arg Gln Glu Met Ser Lys Asp100 105 110Leu Glu Glu Val Lys Gln Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln115 120 125Lys Lys Trp Gln Glu Glu Val Glu Leu Tyr Arg Gln Lys Val Ala Pro130 135 140Leu Gly Ser Glu Leu Arg Glu Gly Ala Arg Gln Lys Leu Gln Glu Leu145 150 155 160Gln Glu Lys Leu Ser Pro Leu Ala Glu Glu Leu Arg Asp Arg Ala Arg165 170 175Thr His Val Asp Ala Leu Arg Ala Gln Leu Ala Pro Tyr Ser Asp Asp180 185 190Leu Arg Glu Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Gly Gly195 200 205Gly Ala Ser Leu Ala Glu Tyr His Ala Arg Ala Ser Glu Gln Leu Ser210 215 220Ala Leu Gly Glu Lys Ala Arg Pro Ala Leu Glu Asp Leu Arg Gln Gly225 230 235 240Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Leu Leu Ala Ala Ile245 250 255Asp Glu Ala Thr Lys Lys Leu Asn Ala Gln260 26520266PRTOryctolagus cuniculus 20Met Lys Ala Val Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser1 5 10 15Gln Ala Arg His Phe Trp Gln Arg Asp Glu Pro Arg Ser Ser Trp Asp20 25 30Lys Ile Lys Asp Phe Ala Thr Val Tyr Val Asp Thr Val Lys Asp Ser35 40 45Gly Arg Glu Tyr Val Ala Gln Phe Glu Ala Ser Ala Phe Gly Lys Gln50 55 60Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Leu Ser Ser Thr Val65 70 75 80Ser Lys Leu Gln Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp85 90 95Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Glu Glu Met Asn Lys Asp100 105 110Leu Gln Glu Val Arg Gln Lys Val Gln Pro Tyr Leu Asp Glu Phe Gln115 120 125Lys Lys Trp Gln Glu Glu Val Glu Arg Tyr Arg Gln Lys Val Glu Pro130 135 140Leu Gly Ala Glu Leu Arg Glu Ser Ala Arg Gln Lys Leu Thr Glu Leu145 150 155 160Gln Glu Lys Leu Ser Pro Leu Ala Glu Glu Leu Arg Asp Ser Ala Arg165 170 175Thr His Val Asp Thr Leu Arg Thr Lys Leu Ala Pro Tyr Ser Asn Glu180 185 190Leu Gln Gln Arg Leu Ala Ala Arg Leu Glu Ser Ile Lys Glu Gly Gly195

200 205Gly Ala Ser Leu Ala Glu Tyr Gln Ala Lys Ala Arg Glu His Leu Ser210 215 220Val Leu Ser Glu Lys Ala Arg Pro Ala Leu Glu Asp Leu Arg Gln Gly225 230 235 240Leu Leu Pro Val Leu Glu Ser Phe Lys Ala Ser Val Gln Asn Val Leu245 250 255Asp Glu Ala Thr Lys Lys Leu Asn Thr Gln260 26521265PRTTupaia glis belangeri 21Met Lys Ala Val Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser1 5 10 15Gln Ala Arg His Phe Trp Gln Gln Asp Glu Pro Gln Ser Ser Trp Asp20 25 30Arg Val Arg Asp Leu Ala Asn Val Tyr Val Asp Ala Val Lys Glu Ser35 40 45Gly Arg Glu Tyr Val Ser Gln Leu Glu Ala Ser Ala Leu Gly Lys Gln50 55 60Leu Asn Leu Lys Leu Val Asp Asn Trp Asp Thr Leu Gly Ser Thr Phe65 70 75 80Gln Lys Val His Glu His Leu Gly Pro Val Ala Gln Glu Phe Trp Glu85 90 95Lys Leu Glu Lys Glu Thr Glu Glu Leu Arg Arg Glu Ile Asn Lys Asp100 105 110Leu Glu Asp Val Arg Gln Lys Thr Gln Pro Phe Leu Asp Glu Ile Gln115 120 125Lys Lys Trp Gln Glu Asp Leu Glu Arg Tyr Arg Gln Lys Val Glu Pro130 135 140Leu Ser Ala Gln Leu Arg Glu Gly Ala Arg Gln Lys Leu Met Glu Leu145 150 155 160Gln Glu Gln Val Thr Pro Leu Gly Glu Asp Leu Arg Asp Ser Val Arg165 170 175Ala Tyr Ala Asp Thr Leu Arg Thr Gln Leu Ala Pro Tyr Ser Glu Gln180 185 190Met Arg Lys Thr Leu Gly Ala Arg Leu Glu Ala Ile Lys Glu Gly Gly195 200 205Ser Ala Ser Leu Ala Glu Tyr His Ala Lys Ala Ser Glu Gln Leu Ser210 215 220Ala Leu Gly Glu Lys Ala Lys Pro Val Leu Glu Asp Ile His Gln Gly225 230 235 240Leu Met Pro Met Trp Glu Ser Phe Lys Thr Gly Val Leu Asn Val Ile245 250 255Asp Glu Ala Ala Lys Lys Leu Thr Ala260 26522264PRTMus musculus 22Met Lys Ala Val Val Leu Ala Val Ala Leu Val Phe Leu Thr Gly Ser1 5 10 15Gln Ala Trp His Val Trp Gln Gln Asp Glu Pro Gln Ser Gln Trp Asp20 25 30Lys Val Lys Asp Phe Ala Asn Val Tyr Val Asp Ala Val Lys Asp Ser35 40 45Gly Arg Asp Tyr Val Ser Gln Phe Glu Ser Ser Ser Leu Gly Gln Gln50 55 60Leu Asn Leu Asn Leu Leu Glu Asn Trp Asp Thr Leu Gly Ser Thr Val65 70 75 80Ser Gln Leu Gln Glu Arg Leu Gly Pro Leu Thr Arg Asp Phe Trp Asp85 90 95Asn Leu Glu Lys Glu Thr Asp Trp Val Arg Gln Glu Met Asn Lys Asp100 105 110Leu Glu Glu Val Lys Gln Lys Val Gln Pro Tyr Leu Asp Glu Phe Gln115 120 125Lys Lys Trp Lys Glu Asp Val Glu Leu Tyr Arg Gln Lys Val Ala Pro130 135 140Leu Gly Ala Glu Leu Gln Glu Ser Ala Arg Gln Lys Leu Gln Glu Leu145 150 155 160Gln Gly Arg Leu Ser Pro Val Ala Glu Glu Phe Arg Asp Arg Met Arg165 170 175Thr His Val Asp Ser Leu Arg Thr Gln Leu Ala Pro His Ser Glu Gln180 185 190Met Arg Glu Ser Leu Ala Gln Arg Leu Ala Glu Leu Lys Ser Asn Pro195 200 205Thr Leu Asn Glu Tyr His Thr Arg Ala Lys Thr His Leu Lys Thr Leu210 215 220Gly Glu Lys Ala Arg Pro Ala Leu Glu Asp Leu Arg His Ser Leu Met225 230 235 240Pro Met Leu Glu Thr Leu Lys Thr Lys Ala Gln Ser Val Ile Asp Lys245 250 255Ala Ser Glu Thr Leu Thr Ala Gln26023259PRTRattus norvegicus 23Met Lys Ala Ala Val Leu Ala Val Ala Leu Val Phe Leu Thr Gly Cys1 5 10 15Gln Ala Trp Glu Phe Trp Gln Gln Asp Glu Pro Gln Ser Gln Trp Asp20 25 30Arg Val Lys Asp Phe Ala Thr Val Tyr Val Asp Ala Val Lys Asp Ser35 40 45Gly Arg Asp Tyr Val Ser Gln Phe Glu Ser Ser Thr Leu Gly Lys Gln50 55 60Leu Asn Leu Asn Leu Leu Asp Asn Trp Asp Thr Leu Gly Ser Thr Val65 70 75 80Gly Arg Leu Gln Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Ala85 90 95Asn Leu Glu Lys Glu Thr Asp Trp Leu Arg Asn Glu Met Asn Lys Asp100 105 110Leu Glu Asn Val Lys Gln Lys Met Gln Pro His Leu Asp Glu Phe Gln115 120 125Glu Lys Trp Asn Glu Glu Val Glu Ala Tyr Arg Gln Lys Leu Glu Pro130 135 140Leu Gly Thr Glu Leu His Lys Asn Ala Lys Glu Met Gln Arg His Leu145 150 155 160Lys Val Val Ala Glu Glu Phe Arg Asp Arg Met Arg Val Asn Ala Asp165 170 175Ala Leu Arg Ala Lys Phe Gly Leu Tyr Ser Asp Gln Met Arg Glu Asn180 185 190Leu Ala Gln Arg Leu Thr Glu Ile Arg Asn His Pro Thr Leu Ile Glu195 200 205Tyr His Thr Lys Ala Gly Asp His Leu Arg Thr Leu Gly Glu Lys Ala210 215 220Lys Pro Ala Leu Asp Asp Leu Gly Gln Gly Leu Met Pro Val Leu Glu225 230 235 240Ala Trp Lys Ala Lys Ile Met Ser Met Ile Asp Glu Ala Lys Lys Lys245 250 255Leu Asn Ala24241PRTErinaceus europaeus 24Asp Glu Ala Lys Ser Tyr Trp Asp Gln Ile Lys Asp Met Leu Thr Val1 5 10 15Tyr Val Asp Thr Ala Lys Asp Ser Gly Lys Asp Tyr Leu Thr Ser Leu20 25 30Asp Thr Ser Ala Leu Gly Gln Gln Leu Asn Lys Lys Leu Ala Asp Asn35 40 45Trp Asp Thr Val Ser Ser Ala Leu Leu Lys Ala Arg Glu Gln Met Lys50 55 60Pro Ile Ala Met Glu Phe Trp Gly Asn Leu Glu Lys Asp Thr Glu Gly65 70 75 80Leu Arg Gln Thr Val Ser Lys Asp Leu Glu Leu Val Lys Glu Lys Val85 90 95Gln Pro Tyr Leu Asp Ser Phe Gln Lys Lys Val Glu Glu Glu Leu Glu100 105 110Leu Tyr Arg Gln Lys Val Ala Pro Leu Ser Ala Glu Trp Arg Glu Gln115 120 125Ala Arg Gln Lys Ala Gln Glu Leu Gln Gln Lys Ala Gly Glu Leu Gly130 135 140Gln Gln His Arg Asp Arg Val Arg Thr His Val Asp Ala Leu Arg Thr145 150 155 160Asp Leu Ala Pro Tyr Gly Glu Glu Ala Arg Lys Leu Leu Leu Gln Arg165 170 175Leu Gln Asp Ile Lys Ala Lys Ser Gly Asp Leu Ala Glu Tyr Gln Thr180 185 190Lys Leu Ser Glu His Leu Lys Ser Phe Gly Glu Lys Ala Gln Pro Thr195 200 205Leu Gln Asp Leu Arg His Gly Leu Glu Pro Leu Trp Glu Gly Ile Lys210 215 220Ala Gly Ala Met Ser Met Leu Glu Glu Leu Gly Lys Lys Leu Asn Ser225 230 235 240Gln25264PRTGallus gallus 25Met Arg Gly Val Leu Val Thr Leu Ala Val Leu Phe Leu Thr Gly Thr1 5 10 15Gln Ala Arg Ser Phe Trp Gln His Asp Glu Pro Gln Thr Pro Leu Asp20 25 30Arg Ile Arg Asp Met Val Asp Val Tyr Leu Glu Thr Val Lys Ala Ser35 40 45Gly Lys Asp Ala Ile Ala Gln Phe Glu Ser Ser Ala Val Gly Lys Gln50 55 60Leu Asp Leu Lys Leu Ala Asp Asn Leu Asp Thr Leu Ser Ala Ala Ala65 70 75 80Ala Lys Leu Arg Glu Asp Met Ala Pro Tyr Tyr Lys Glu Val Arg Glu85 90 95Met Trp Leu Lys Asp Thr Glu Ala Leu Arg Ala Glu Leu Thr Lys Asp100 105 110Leu Glu Glu Val Lys Glu Lys Ile Arg Pro Phe Leu Asp Gln Phe Ser115 120 125Ala Lys Trp Thr Glu Glu Leu Glu Gln Tyr Arg Gln Arg Leu Thr Pro130 135 140Val Ala Gln Glu Leu Lys Glu Leu Thr Lys Gln Lys Val Glu Leu Met145 150 155 160Gln Ala Lys Leu Thr Pro Val Ala Glu Glu Ala Arg Asp Arg Leu Arg165 170 175Gly His Val Glu Glu Leu Arg Lys Asn Leu Ala Pro Tyr Ser Asp Glu180 185 190Leu Arg Gln Lys Leu Ser Gln Lys Leu Glu Glu Ile Arg Glu Lys Gly195 200 205Ile Pro Gln Ala Ser Glu Tyr Gln Ala Lys Val Met Glu Gln Leu Ser210 215 220Asn Leu Arg Glu Lys Met Thr Pro Leu Val Gln Glu Phe Arg Glu Arg225 230 235 240Leu Thr Pro Tyr Ala Glu Asn Leu Lys Asn Arg Leu Ile Ser Phe Leu245 250 255Asp Glu Leu Gln Lys Ser Val Ala26026264PRTCoturnix coturnix japonica 26Met Arg Gly Val Leu Val Thr Leu Ala Val Leu Phe Leu Thr Gly Thr1 5 10 15Gln Ala Arg Ser Phe Trp Gln His Asp Asp Pro Gln Thr Pro Leu Asp20 25 30Arg Ile Arg Asp Met Leu Asp Val Tyr Leu Glu Thr Val Lys Ala Ser35 40 45Gly Lys Asp Ala Ile Ser Gln Phe Glu Ser Ser Ala Val Gly Lys Gln50 55 60Leu Asp Leu Lys Leu Ala Asp Asn Leu Asp Thr Leu Ser Ala Ala Ala65 70 75 80Ala Lys Leu Arg Glu Asp Met Thr Pro Tyr Tyr Arg Glu Val Arg Glu85 90 95Met Trp Leu Lys Asp Thr Glu Ala Leu Arg Ala Glu Leu Thr Lys Asp100 105 110Leu Glu Glu Val Lys Glu Lys Ile Arg Pro Phe Leu Asp Gln Phe Ser115 120 125Ala Lys Trp Thr Glu Glu Val Glu Gln Tyr Arg Gln Arg Leu Ala Pro130 135 140Val Ala Gln Glu Leu Lys Asp Leu Thr Lys Gln Lys Val Glu Leu Met145 150 155 160Gln Ala Lys Leu Thr Pro Val Ala Glu Glu Val Arg Asp Arg Leu Arg165 170 175Glu Gln Val Glu Glu Leu Arg Lys Asn Leu Ala Pro Tyr Ser Ser Glu180 185 190Leu Arg Gln Lys Leu Ser Gln Lys Leu Glu Glu Ile Arg Glu Arg Gly195 200 205Ile Pro Gln Ala Ser Glu Tyr Gln Ala Lys Val Val Glu Gln Leu Ser210 215 220Asn Leu Arg Glu Lys Met Thr Pro Leu Val Gln Glu Phe Lys Glu Arg225 230 235 240Leu Thr Pro Tyr Ala Glu Asn Leu Lys Asn Arg Leu Ile Asp Leu Leu245 250 255Asp Glu Val Gln Lys Thr Met Ala26027264PRTAnas platyrhynchos 27Met Arg Val Val Val Val Thr Leu Ala Leu Leu Phe Leu Thr Gly Thr1 5 10 15Gln Ala Arg Tyr Phe Trp Gln His Asp Glu Pro Gln Ala Pro Leu Asp20 25 30Arg Leu Arg Asp Leu Val Asp Val Tyr Leu Glu Thr Val Lys Ala Ser35 40 45Gly Lys Asp Ala Ile Ala Gln Phe Glu Ala Ser Ala Val Gly Lys Gln50 55 60Leu Asp Leu Lys Leu Ala Asp Asn Leu Asp Thr Leu Gly Ala Ala Ala65 70 75 80Ala Lys Leu Arg Glu Asp Met Ala Pro Tyr Tyr Lys Glu Val Arg Glu85 90 95Met Trp Leu Lys Asp Thr Glu Ser Leu Arg Ala Glu Leu Thr Lys Asp100 105 110Leu Glu Glu Val Lys Glu Lys Ile Arg Pro Phe Leu Asp Gln Phe Ser115 120 125Ala Lys Trp Thr Glu Glu Leu Glu Gln Tyr Arg Gln Arg Leu Ala Pro130 135 140Val Ala Glu Glu Leu Lys Glu Leu Thr Lys Gln Lys Val Glu Leu Met145 150 155 160Gln Gln Lys Leu Thr Pro Val Ala Glu Glu Ala Arg Asp Arg Leu Arg165 170 175Gly His Val Glu Glu Leu Arg Lys Asn Leu Ala Pro Tyr Ser Asp Glu180 185 190Leu Arg Gln Lys Leu Ser Gln Lys Leu Glu Glu Ile Arg Glu Lys Gly195 200 205Ile Pro Gln Ala Ala Glu Tyr Gln Ala Lys Val Val Glu Gln Leu Ser210 215 220Asn Leu Arg Glu Lys Met Thr Pro Leu Val Gln Asp Phe Lys Glu Arg225 230 235 240Leu Thr Pro Tyr Ala Glu Asn Leu Lys Thr Arg Phe Ile Ser Leu Leu245 250 255Asp Glu Leu Gln Lys Thr Val Ala26028262PRTOncorhynchus mykiss 28Met Lys Phe Leu Ala Leu Ala Leu Thr Ile Leu Leu Ala Ala Gly Thr1 5 10 15Gln Ala Phe Pro Met Gln Ala Asp Ala Pro Ser Gln Leu Glu His Val20 25 30Lys Ala Ala Leu Ser Met Tyr Ile Ala Gln Val Lys Leu Thr Ala Gln35 40 45Arg Ser Ile Asp Leu Leu Asp Asp Thr Glu Tyr Lys Glu Tyr Lys Met50 55 60Gln Leu Thr Gln Ser Leu Asp Asn Leu Gln Gln Tyr Ala Asp Ala Thr65 70 75 80Ser Gln Ser Leu Ala Pro Tyr Ser Glu Ala Phe Gly Thr Gln Leu Thr85 90 95Asp Ala Thr Ala Ala Val Arg Ala Glu Val Met Lys Asp Val Glu Glu100 105 110Leu Arg Ser Gln Leu Glu Pro Lys Arg Ala Glu Leu Lys Glu Val Leu115 120 125Asp Lys His Ile Asp Glu Tyr Arg Lys Lys Leu Glu Pro Leu Ile Lys130 135 140Glu His Ile Glu Leu Arg Arg Thr Glu Met Glu Ala Phe Arg Ala Lys145 150 155 160Met Glu Pro Ile Val Glu Glu Leu Arg Ala Lys Val Ala Ile Asn Val165 170 175Glu Glu Thr Lys Thr Lys Leu Met Pro Ile Val Glu Ile Val Arg Ala180 185 190Lys Leu Thr Glu Arg Leu Glu Glu Leu Arg Thr Leu Ala Ala Pro Tyr195 200 205Ala Glu Glu Tyr Lys Glu Gln Met Ile Lys Ala Val Gly Glu Val Arg210 215 220Glu Lys Val Ser Pro Leu Ser Glu Asp Phe Lys Gly Gln Val Gly Pro225 230 235 240Ala Ala Glu Gln Ala Lys Gln Lys Leu Leu Ala Phe Tyr Glu Thr Ile245 250 255Ser Gln Ala Met Lys Ala26029262PRTSalmo trutta 29Met Lys Phe Leu Ala Leu Ala Leu Thr Ile Leu Leu Ala Ala Ala Thr1 5 10 15Gln Ala Val Pro Met Gln Ala Asp Ala Pro Ser Gln Leu Glu His Val20 25 30Lys Val Ala Met Met Glu Tyr Met Ala Gln Val Lys Glu Thr Gly Gln35 40 45Arg Ser Ile Asp Leu Leu Asp Asp Thr Glu Phe Lys Glu Tyr Lys Val50 55 60Gln Leu Ser Gln Ser Leu Asp Asn Leu Gln Gln Tyr Ala Gln Thr Thr65 70 75 80Ser Gln Ser Leu Ala Pro Tyr Ser Glu Ala Phe Gly Ala Gln Leu Thr85 90 95Asp Ala Ala Ala Ala Val Arg Ala Glu Val Met Lys Asp Val Glu Asp100 105 110Val Arg Thr Gln Leu Glu Pro Lys Arg Ala Glu Leu Lys Glu Val Leu115 120 125Asp Lys His Ile Asp Glu Tyr Arg Lys Lys Leu Glu Pro Leu Ile Lys130 135 140Glu Ile Val Glu Gln Arg Arg Thr Glu Leu Glu Ala Phe Arg Val Lys145 150 155 160Met Glu Pro Val Val Glu Glu Met Arg Ala Lys Val Ser Thr Asn Val165 170 175Glu Glu Thr Lys Ala Lys Leu Met Pro Ile Val Glu Thr Val Arg Ala180 185 190Lys Leu Thr Glu Arg Leu Glu Glu Leu Arg Thr Leu Ala Ala Pro Tyr195 200 205Ala Glu Glu Tyr Lys Glu Gln Met Phe Lys Ala Val Gly Glu Val Arg210 215 220Glu Lys Val Gly Pro Leu Thr Asn Asp Phe Lys Gly Gln Val Gly Pro225 230 235 240Ala Ala Glu Gln Ala Lys Glu Lys Leu Met Asp Phe Tyr Glu Thr Ile245 250 255Ser Gln Ala Met Lys Ala26030258PRTSalmo salar 30Met Lys Phe Leu Val Leu Ala Leu Thr Ile Leu Leu Ala Ala Gly Thr1 5 10 15Gln Ala Phe Pro Met Gln Ala Asp Ala Pro Ser Gln Leu Glu His Val20 25 30Lys Ala Ala Leu Asn Met Tyr Ile Ala Gln Val Lys Leu Thr Ala Gln35 40 45Arg Ser Ile Asp Leu Leu Asp Asp Thr Glu Tyr Lys Glu Tyr Lys Met50 55 60Gln Leu Ser Gln Ser Leu Asp Asn Leu Gln Gln Phe Ala Asp Ser Thr65 70 75 80Ser Lys Ser Trp Pro Pro Thr Pro Arg Ser Ser Ala Pro Ser Cys Asp85 90 95Ala Thr Ala Thr Val Arg Ala Glu Val Met Lys Asp Val Glu Asp Val100 105 110Arg Thr Gln Leu Glu Pro Lys Arg Ala Glu Leu Thr Glu Val Leu Asn115 120 125Lys His Ile Asp Glu Tyr Arg Lys Lys Leu Glu Pro Leu Ile Lys Gln130 135 140His Ile Glu Leu Arg Arg Thr Glu Met Asp Ala Phe Arg Ala Lys Ile145 150 155 160Asp Pro Val Val Glu Glu Met Arg Ala Lys Val Ala Val Asn Val Glu165 170 175Glu Thr Lys Thr Lys Leu Met Pro Ile Val Glu Ile Val Arg Ala Lys180 185 190Leu Thr Glu Arg Leu Glu Glu Leu Arg Thr Leu Ala Ala Pro Tyr Ala195 200 205Glu Glu Tyr Lys Glu Gln Met Phe Lys Ala Val Gly Glu Val Arg Glu210

215 220Lys Val Ala Pro Leu Ser Glu Asp Phe Lys Ala Arg Trp Ala Pro Pro225 230 235 240Pro Arg Arg Pro Ser Lys Ser Ser Trp Leu Ser Thr Arg Pro Ser Ala245 250 255Arg Pro31262PRTBrachydanio rerio 31Met Lys Phe Val Ala Leu Ala Leu Thr Leu Leu Leu Ala Leu Gly Ser1 5 10 15Gln Ala Asn Leu Phe Gln Ala Asp Ala Pro Thr Gln Leu Glu His Tyr20 25 30Lys Ala Ala Ala Leu Val Tyr Leu Asn Gln Val Lys Asp Gln Ala Glu35 40 45Lys Ala Leu Asp Asn Leu Asp Gly Thr Asp Tyr Glu Gln Tyr Lys Leu50 55 60Gln Leu Ser Glu Ser Leu Thr Lys Leu Gln Glu Tyr Ala Gln Thr Thr65 70 75 80Ser Gln Ala Leu Thr Pro Tyr Ala Glu Thr Ile Ser Thr Gln Leu Met85 90 95Glu Asn Thr Lys Gln Leu Arg Glu Arg Val Met Thr Asp Val Glu Asp100 105 110Leu Arg Ser Lys Leu Glu Pro His Arg Ala Glu Leu Tyr Thr Ala Leu115 120 125Gln Lys His Ile Asp Glu Tyr Arg Glu Lys Leu Glu Pro Val Phe Gln130 135 140Glu Tyr Ser Ala Leu Asn Arg Gln Asn Ala Glu Gln Leu Arg Ala Lys145 150 155 160Leu Glu Pro Leu Met Asp Asp Ile Arg Lys Ala Phe Glu Ser Asn Ile165 170 175Glu Glu Thr Lys Ser Lys Val Val Pro Met Val Glu Ala Val Arg Thr180 185 190Lys Leu Thr Glu Arg Leu Glu Asp Leu Arg Thr Met Ala Ala Pro Tyr195 200 205Ala Glu Glu Tyr Lys Glu Gln Leu Val Lys Ala Val Glu Glu Ala Arg210 215 220Glu Lys Ile Ala Pro His Thr Gln Asp Leu Gln Thr Arg Met Glu Pro225 230 235 240Tyr Met Glu Asn Val Arg Thr Thr Phe Ala Gln Met Tyr Glu Thr Ile245 250 255Ala Lys Ala Ile Gln Ala26032260PRTSparus aurata 32Met Lys Phe Ala Ala Leu Ala Leu Ala Leu Leu Leu Ala Val Gly Ser1 5 10 15His Ala Ala Ser Met Gln Ala Asp Ala Pro Ser Gln Leu Asp His Ala20 25 30Arg Ala Val Leu Asp Val Tyr Leu Thr Gln Val Lys Asp Met Ser Leu35 40 45Arg Ala Val Asn Gln Leu Asp Asp Pro Gln Tyr Ala Glu Phe Lys Thr50 55 60Asn Leu Ala Gln Arg Ile Glu Glu Met Tyr Thr Gln Ile Lys Thr Leu65 70 75 80Gln Gly Ser Val Ser Pro Met Thr Asp Ser Phe Tyr Asn Thr Val Met85 90 95Glu Val Thr Lys Asp Thr Arg Glu Ser Leu Asn Val Asp Leu Glu Ala100 105 110Leu Lys Ser Ser Leu Ala Pro Gln Asn Glu Gln Leu Lys Gln Val Ile115 120 125Glu Lys His Leu Asn Asp Tyr Arg Thr Leu Leu Thr Pro Ile Tyr Asn130 135 140Asp Tyr Lys Thr Lys His Asp Glu Glu Met Ala Ala Leu Lys Thr Arg145 150 155 160Leu Glu Pro Val Met Glu Glu Leu Arg Thr Lys Ile Gln Ala Asn Val165 170 175Glu Glu Thr Lys Ala Val Leu Met Pro Met Val Glu Thr Val Arg Thr180 185 190Lys Val Thr Glu Arg Leu Glu Ser Leu Arg Glu Val Val Gln Pro Tyr195 200 205Val Gln Glu Tyr Lys Glu Gln Met Lys Gln Met Tyr Asp Gln Ala Gln210 215 220Thr Val Asp Thr Asp Ala Leu Arg Thr Lys Ile Thr Pro Leu Val Glu225 230 235 240Glu Ile Lys Val Lys Met Asn Ala Ile Phe Glu Ile Ile Ala Ala Ser245 250 255Val Thr Lys Ser26033396PRTHomo sapiens 33Met Phe Leu Lys Ala Val Val Leu Thr Leu Ala Leu Val Ala Val Ala1 5 10 15Gly Ala Arg Ala Glu Val Ser Ala Asp Gln Val Ala Thr Val Met Trp20 25 30Asp Tyr Phe Ser Gln Leu Ser Asn Asn Ala Lys Glu Ala Val Glu His35 40 45Leu Gln Lys Ser Glu Leu Thr Gln Gln Leu Asn Ala Leu Phe Gln Asp50 55 60Lys Leu Gly Glu Val Asn Thr Tyr Ala Gly Asp Leu Gln Lys Lys Leu65 70 75 80Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Ala Lys Asp Ser Glu85 90 95Lys Leu Lys Glu Glu Ile Gly Lys Glu Leu Glu Glu Leu Arg Ala Arg100 105 110Leu Leu Pro His Ala Asn Glu Val Ser Gln Lys Ile Gly Asp Asn Leu115 120 125Arg Glu Leu Gln Gln Arg Leu Glu Pro Tyr Ala Asp Gln Leu Arg Thr130 135 140Gln Val Asn Thr Gln Ala Glu Gln Leu Arg Arg Gln Leu Thr Pro Tyr145 150 155 160Ala Gln Arg Met Glu Arg Val Leu Arg Glu Asn Ala Asp Ser Leu Gln165 170 175Ala Ser Leu Arg Pro His Ala Asp Glu Leu Lys Ala Lys Ile Asp Gln180 185 190Asn Val Glu Glu Leu Lys Gly Arg Leu Thr Pro Tyr Ala Asp Glu Phe195 200 205Lys Val Lys Ile Asp Gln Thr Val Glu Glu Leu Arg Arg Ser Leu Ala210 215 220Pro Tyr Ala Gln Asp Thr Gln Glu Lys Leu Asn His Gln Leu Glu Gly225 230 235 240Leu Thr Phe Gln Met Lys Lys Asn Ala Glu Glu Leu Lys Ala Arg Ile245 250 255Ser Ala Ser Ala Glu Glu Leu Arg Gln Arg Leu Ala Pro Leu Ala Glu260 265 270Asp Val Arg Gly Asn Leu Lys Gly Asn Thr Glu Gly Leu Gln Lys Ser275 280 285Leu Ala Glu Leu Gly Gly His Leu Asp Gln Gln Val Glu Glu Phe Arg290 295 300Arg Arg Val Glu Pro Tyr Gly Glu Asn Phe Asn Lys Ala Leu Val Gln305 310 315 320Gln Met Glu Gln Leu Arg Gln Lys Leu Gly Pro His Ala Gly Asp Val325 330 335Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp Lys Val Asn340 345 350Ser Phe Phe Ser Thr Phe Lys Glu Lys Glu Ser Gln Asp Lys Thr Leu355 360 365Ser Leu Pro Glu Leu Glu Gln Gln Gln Glu Gln Gln Gln Glu Gln Gln370 375 380Gln Glu Gln Val Gln Met Leu Ala Pro Leu Glu Ser385 390 39534429PRTMacaca fascicularis 34Met Phe Leu Lys Ala Val Val Leu Thr Leu Ala Leu Val Ala Val Thr1 5 10 15Gly Ala Arg Ala Glu Val Ser Ala Asp Gln Val Ala Thr Val Met Trp20 25 30Asp Tyr Phe Ser Gln Leu Ser Ser Asn Ala Lys Glu Ala Val Glu His35 40 45Leu Gln Lys Ser Glu Leu Thr Gln Gln Leu Asn Ala Leu Phe Gln Asp50 55 60Lys Leu Gly Glu Val Asn Thr Tyr Ala Gly Asp Leu Gln Lys Lys Leu65 70 75 80Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Ala Lys Asp Ser Glu85 90 95Lys Leu Lys Glu Glu Ile Arg Lys Glu Leu Glu Glu Val Arg Ala Arg100 105 110Leu Leu Pro His Ala Asn Glu Val Ser Gln Lys Ile Gly Glu Asn Val115 120 125Arg Glu Leu Gln Gln Arg Leu Glu Pro Tyr Thr Asp Gln Leu Arg Thr130 135 140Gln Val Asn Thr Gln Thr Glu Gln Leu Arg Arg Gln Leu Thr Pro Tyr145 150 155 160Ala Gln Arg Met Glu Arg Val Leu Arg Glu Asn Ala Asp Ser Leu Gln165 170 175Thr Ser Leu Arg Pro His Ala Asp Gln Leu Lys Ala Lys Ile Asp Gln180 185 190Asn Val Glu Glu Leu Lys Glu Arg Leu Thr Pro Tyr Ala Asp Glu Phe195 200 205Lys Val Lys Ile Asp Gln Thr Val Glu Glu Leu Arg Arg Ser Leu Ala210 215 220Pro Tyr Ala Gln Asp Ala Gln Glu Lys Leu Asn His Gln Leu Glu Gly225 230 235 240Leu Ala Phe Gln Met Lys Lys Asn Ala Glu Glu Leu Lys Ala Arg Ile245 250 255Ser Ala Ser Ala Glu Glu Leu Arg Gln Arg Leu Ala Pro Leu Ala Glu260 265 270Asp Met Arg Gly Asn Leu Arg Gly Asn Thr Glu Gly Leu Gln Lys Ser275 280 285Leu Ala Glu Leu Gly Gly His Leu Asp Arg His Val Glu Glu Phe Arg290 295 300Leu Arg Val Glu Pro Tyr Gly Glu Asn Phe Asn Lys Ala Leu Val Gln305 310 315 320Gln Met Glu Gln Leu Arg Gln Lys Leu Gly Pro His Ala Gly Asp Val325 330 335Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp Lys Val Asn340 345 350Ser Phe Phe Ser Thr Phe Lys Glu Lys Glu Ser Gln Asp Asn Thr Leu355 360 365Ser Leu Pro Glu Pro Glu Gln Gln Arg Glu Gln Gln Gln Glu Gln Gln370 375 380Gln Glu Gln Glu Gln Glu Gln Gln Gln Gln Gln Glu Gln Gln Gln Gln385 390 395 400Gln Glu Gln Gln Arg Glu Gln Gln Gln Gln Glu Gln Gln Gln Glu Gln405 410 415Gln Gln Glu Gln Val Gln Met Leu Ala Pro Leu Glu Ser420 42535395PRTMus musculus 35Met Phe Leu Lys Ala Ala Val Leu Thr Leu Ala Leu Val Ala Ile Thr1 5 10 15Gly Thr Arg Ala Glu Val Thr Ser Asp Gln Val Ala Asn Val Val Trp20 25 30Asp Tyr Phe Thr Gln Leu Ser Asn Asn Ala Lys Glu Ala Val Glu Gln35 40 45Phe Gln Lys Thr Asp Val Thr Gln Gln Leu Ser Thr Leu Phe Gln Asp50 55 60Lys Leu Gly Asp Ala Ser Thr Tyr Ala Asp Gly Val His Asn Lys Leu65 70 75 80Val Pro Phe Val Val Gln Leu Ser Gly His Leu Ala Lys Glu Thr Glu85 90 95Arg Val Lys Glu Glu Ile Lys Lys Glu Leu Glu Asp Leu Arg Asp Arg100 105 110Met Met Pro His Ala Asn Lys Val Thr Gln Thr Phe Gly Glu Asn Met115 120 125Gln Lys Leu Gln Glu His Leu Lys Pro Tyr Ala Val Asp Leu Gln Asp130 135 140Gln Ile Asn Thr Gln Thr Gln Glu Met Lys Leu Gln Leu Thr Pro Tyr145 150 155 160Ile Gln Arg Met Gln Thr Thr Ile Lys Glu Asn Val Asp Asn Leu His165 170 175Thr Ser Met Met Pro Leu Ala Thr Asn Leu Lys Asp Lys Phe Asn Arg180 185 190Asn Met Glu Glu Leu Lys Gly His Leu Thr Pro Arg Ala Asn Glu Leu195 200 205Lys Ala Thr Ile Asp Gln Asn Leu Glu Asp Leu Arg Arg Ser Leu Ala210 215 220Pro Leu Thr Val Gly Val Gln Glu Lys Leu Asn His Gln Met Glu Gly225 230 235 240Leu Ala Phe Gln Met Lys Lys Asn Ala Glu Glu Leu Gln Thr Lys Val245 250 255Ser Ala Lys Ile Asp Gln Leu Gln Lys Asn Leu Ala Pro Leu Val Glu260 265 270Asp Val Gln Ser Lys Val Lys Gly Asn Thr Glu Gly Leu Gln Lys Ser275 280 285Leu Glu Asp Leu Asn Arg Gln Leu Glu Gln Gln Val Glu Glu Phe Arg290 295 300Arg Thr Val Glu Pro Met Gly Glu Met Phe Asn Lys Ala Leu Val Gln305 310 315 320Gln Leu Glu Gln Phe Arg Gln Gln Leu Gly Pro Asn Ser Gly Glu Val325 330 335Glu Ser His Leu Ser Phe Leu Glu Lys Ser Leu Arg Glu Lys Val Asn340 345 350Ser Phe Met Ser Thr Leu Glu Lys Lys Gly Ser Pro Asp Gln Pro Gln355 360 365Ala Leu Pro Leu Pro Glu Gln Ala Gln Glu Gln Ala Gln Glu Gln Ala370 375 380Gln Glu Gln Val Gln Pro Lys Pro Leu Glu Ser385 390 39536401PRTPapio anubis 36Gly Ala Arg Ala Glu Val Ser Ala Asp Gln Val Ala Thr Val Met Trp1 5 10 15Asp Tyr Phe Ser Gln Leu Ser Ser Asn Ala Lys Glu Ala Val Glu His20 25 30Leu Gln Lys Ser Glu Leu Thr Gln Gln Leu Asn Ala Leu Phe Gln Asp35 40 45Lys Leu Gly Glu Val Asn Thr Tyr Ala Gly Asp Leu Gln Lys Lys Leu50 55 60Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Ala Lys Asp Ser Lys65 70 75 80Lys Leu Lys Glu Glu Ile Arg Lys Glu Leu Glu Glu Val Arg Ala Arg85 90 95Leu Leu Pro His Ala Asn Glu Val Ser Gln Lys Ile Gly Glu Asn Val100 105 110Arg Glu Leu Gln Gln Arg Leu Glu Pro Tyr Thr Asp Gln Leu Arg Thr115 120 125Gln Val Asn Thr Gln Thr Glu Gln Leu Arg Arg Gln Leu Thr Pro Tyr130 135 140Ala Gln Arg Met Glu Arg Val Leu Arg Glu Asn Ala Asp Ser Leu Gln145 150 155 160Thr Ser Leu Arg Pro His Ala Asp Gln Leu Lys Ala Lys Ile Asp Gln165 170 175Asn Val Glu Glu Leu Lys Gly Arg Leu Thr Pro Tyr Ala Asp Glu Phe180 185 190Lys Val Lys Ile Asp Gln Thr Val Glu Glu Leu Arg Arg Ser Leu Ala195 200 205Pro Tyr Ala Gln Asp Ala Gln Glu Lys Leu Asn His Gln Leu Glu Gly210 215 220Leu Ala Phe Gln Met Lys Lys Asn Ala Glu Glu Leu Lys Ala Arg Ile225 230 235 240Ser Ala Ser Ala Glu Glu Leu Arg Gln Arg Leu Ala Pro Leu Ala Glu245 250 255Asp Met Arg Gly Asn Leu Arg Gly Asn Thr Glu Gly Leu Gln Lys Ser260 265 270Leu Ala Glu Leu Gly Gly His Leu Asp Arg His Val Glu Glu Phe Arg275 280 285Leu Arg Val Glu Pro Tyr Gly Glu Asn Phe Asn Lys Ala Leu Val Gln290 295 300Gln Met Glu Gln Leu Arg Gln Lys Leu Gly Pro His Ala Gly Asp Val305 310 315 320Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp Lys Val Asn325 330 335Ser Phe Phe Ser Thr Phe Lys Glu Lys Glu Ser Gln Asp Asn Thr Leu340 345 350Ser Leu Pro Glu Pro Glu Gln Gln Gln Glu Gln Gln Gln Glu Gln Glu355 360 365Gln Gln Gln Glu Gln Gln Glu Glu Gln Gln Gln Gln Glu Gln Gln Gln370 375 380Glu Gln Glu Gln Gln Gln Glu Gln Val Gln Met Leu Ala Pro Leu Glu385 390 395 400Ser37382PRTSus scrofa 37Met Phe Leu Lys Ala Val Val Leu Ser Leu Ala Leu Val Ala Val Thr1 5 10 15Gly Ala Arg Ala Glu Val Asn Ala Asp Gln Val Ala Thr Val Met Trp20 25 30Asp Tyr Phe Ser Gln Leu Gly Ser Asn Ala Lys Lys Ala Val Glu His35 40 45Leu Gln Lys Ser Glu Leu Thr Gln Gln Leu Asn Thr Leu Phe Gln Asp50 55 60Lys Leu Gly Glu Val Asn Thr Tyr Thr Glu Asp Leu Gln Lys Lys Leu65 70 75 80Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Thr Lys Asp Ser Glu85 90 95Lys Leu Lys Glu Glu Ile Arg Arg Glu Leu Glu Glu Leu Arg Ala Arg100 105 110Leu Leu Pro His Ala Thr Glu Val Ser Gln Lys Ile Gly Asp Asn Val115 120 125Arg Glu Leu Gln Gln Arg Leu Gly Pro Phe Thr Gly Gly Leu Arg Thr130 135 140Gln Val Asn Thr Gln Val Gln Gln Leu Gln Arg Gln Leu Lys Pro Tyr145 150 155 160Ala Glu Arg Met Glu Ser Val Leu Arg Gln Asn Ile Arg Asn Leu Glu165 170 175Ala Ser Val Ala Pro Tyr Ala Asp Glu Phe Lys Ala Lys Ile Asp Gln180 185 190Asn Val Glu Glu Leu Lys Gly Ser Leu Thr Pro Tyr Ala Glu Glu Leu195 200 205Lys Ala Lys Ile Asp Gln Asn Val Glu Glu Leu Arg Arg Ser Leu Ala210 215 220Pro Tyr Ala Gln Asp Val Gln Glu Lys Leu Asn His Gln Leu Glu Gly225 230 235 240Leu Ala Phe Gln Met Lys Lys Gln Ala Glu Glu Leu Lys Ala Lys Ile245 250 255Ser Ala Asn Ala Asp Glu Leu Arg Gln Lys Leu Val Pro Val Ala Glu260 265 270Asn Val His Gly His Leu Lys Gly Asn Thr Glu Gly Leu Gln Lys Ser275 280 285Leu Leu Glu Leu Arg Ser His Leu Asp Gln Gln Val Glu Glu Phe Arg290 295 300Leu Lys Val Glu Pro Tyr Gly Glu Thr Phe Asn Lys Ala Leu Val Gln305 310 315 320Gln Val Glu Asp Leu Arg Gln Lys Leu Gly Pro Leu Ala Gly Asp Val325 330 335Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp Lys Val Asn340 345 350Thr Phe Phe Ser Thr Leu Lys Glu Glu Ala Ser Gln Gly Gln Ser Gln355 360 365Ala Leu Pro Ala Gln Glu Lys Ala Gln Ala Pro Leu Glu Gly370 375 38038391PRTRattus norvegicus 38Met Phe Leu Lys Ala Val Val Leu Thr Val Ala Leu Val Ala Ile Thr1 5 10 15Gly Thr Gln Ala Glu Val Thr Ser Asp Gln Val Ala Asn Val Met Trp20 25 30Asp Tyr Phe Thr Gln Leu Ser Asn Asn Ala Lys Glu Ala Val Glu Gln35 40 45Leu Gln Lys Thr Asp Val Thr Gln Gln Leu Asn Thr Leu Phe Gln Asp50 55 60Lys Leu Gly Asn Ile Asn Thr Tyr Ala Asp Asp

Leu Gln Asn Lys Leu65 70 75 80Val Pro Phe Ala Val Gln Leu Ser Gly His Leu Thr Lys Glu Thr Glu85 90 95Arg Val Arg Glu Glu Ile Gln Lys Glu Leu Glu Asp Leu Arg Ala Asn100 105 110Met Met Pro His Ala Asn Lys Val Ser Gln Met Phe Gly Asp Asn Val115 120 125Gln Lys Leu Gln Glu His Leu Arg Pro Tyr Ala Thr Asp Leu Gln Ala130 135 140Gln Ile Asn Ala Gln Thr Gln Asp Met Lys Arg Gln Leu Thr Pro Tyr145 150 155 160Ile Gln Arg Met Gln Thr Thr Ile Gln Asp Asn Val Glu Asn Leu Gln165 170 175Ser Ser Met Val Pro Phe Ala Asn Glu Leu Lys Glu Lys Phe Asn Gln180 185 190Asn Met Glu Gly Leu Lys Gly Gln Leu Thr Pro Arg Ala Asn Glu Leu195 200 205Lys Ala Thr Ile Asp Gln Asn Leu Glu Asp Leu Arg Ser Arg Leu Ala210 215 220Pro Leu Ala Glu Gly Val Gln Glu Lys Leu Asn His Gln Met Glu Gly225 230 235 240Leu Ala Phe Gln Met Lys Lys Asn Ala Glu Glu Leu Gln Thr Lys Val245 250 255Ser Thr Asn Ile Asp Gln Leu Gln Lys Asn Leu Ala Pro Leu Val Glu260 265 270Asp Val Gln Ser Lys Leu Lys Gly Asn Thr Glu Gly Leu Gln Lys Ser275 280 285Leu Glu Asp Leu Asn Lys Gln Leu Asp Gln Gln Val Glu Val Phe Arg290 295 300Arg Ala Val Glu Pro Leu Gly Asp Lys Phe Asn Met Ala Leu Val Gln305 310 315 320Gln Met Glu Lys Phe Arg Gln Gln Leu Gly Ser Asp Ser Gly Asp Val325 330 335Glu Ser His Leu Ser Phe Leu Glu Lys Asn Leu Arg Glu Lys Val Ser340 345 350Ser Phe Met Ser Thr Leu Gln Lys Lys Gly Ser Pro Asp Gln Pro Leu355 360 365Ala Leu Pro Leu Pro Glu Gln Val Gln Glu Gln Val Gln Glu Gln Val370 375 380Gln Pro Lys Pro Leu Glu Ser385 3903936PRTHomo sapiens 39Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu Asp Thr1 5 10 15Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu Gln Thr20 25 30Val Cys Leu Lys354036PRTMus musculus 40Leu Val Ser Ser Lys Met Phe Glu Glu Leu Lys Asn Arg Met Asp Val1 5 10 15Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Lys Gln Ala Leu Gln Thr20 25 30Val Cys Leu Lys354136PRTBox taurus 41Arg Arg Val Lys Glu Lys Asp Gly Asp Leu Lys Thr Gln Val Glu Lys1 5 10 15Leu Trp Arg Glu Val Asn Ala Leu Lys Glu Met Gln Ala Leu Gln Thr20 25 30Val Cys Leu Arg354236PRTCarcharhinus springeri 42Ser Lys Ser Gly Lys Gly Lys Asp Asp Leu Arg Asn Glu Ile Asp Lys1 5 10 15Leu Trp Arg Glu Val Asn Ser Leu Lys Glu Met Gln Ala Leu Gln Thr20 25 30Val Cys Leu Lys35431282DNAArtificial SequencepT7 H6UbiFx Apo A-1 plasmid 43gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac gga tca cag atc ttt gtg aag acc ctc act ggc aaa 162His His His His Gly Ser Gln Ile Phe Val Lys Thr Leu Thr Gly Lys10 15 20acc atc acc ctt gag gtc gag ccc agt gac acc att gag aat gtc aaa 210Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys25 30 35gcc aaa att caa gac aag gag ggt atc cca cct gac cag cag cgt ctg 258Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu40 45 50ata ttt gcc ggc aaa cag ctg gaa gat gga cgt act ttg tct gac tac 306Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr55 60 65aat att caa aag gag tct act ctt cat ctt gtg ttg aga ctt cgt ggt 354Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly70 75 80 85gga tcc atc gag ggt agg ggt gga gat gaa ccc ccc cag agc ccc tgg 402Gly Ser Ile Glu Gly Arg Gly Gly Asp Glu Pro Pro Gln Ser Pro Trp90 95 100gat cga gtg aag gac ctg gcc act gtg tac gtg gat gtg ctc aaa gac 450Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp105 110 115agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc gcc ttg gga aaa 498Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys120 125 130cag cta aac cta aag ctc ctt gac aac tgg gac agc gtg acc tcc acc 546Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr135 140 145ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc cag gag ttc tgg 594Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp150 155 160 165gat aac ctg gaa aag gag aca gag ggc ctg agg cag gag atg agc aag 642Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys170 175 180gat ctg gag gag gtg aag gcc aag gtg cag ccc tac ctg gac gac ttc 690Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe185 190 195cag aag aag tgg cag gag gag atg gag ctc tac cgc cag aag gtg gag 738Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu200 205 210ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag aag ctg cac gag 786Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu215 220 225ctg caa gag aag ctg agc cca ctg ggc gag gag atg cgc gac cgc gcg 834Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala230 235 240 245cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc ccc tac agc gac 882Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp250 255 260gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct ctc aag gag aac 930Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn265 270 275ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc acc gag cat ctg 978Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu280 285 290agc acg ctc agc gag aag gcc aag ccc gcg ctc gag gac ctc cgc caa 1026Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln295 300 305ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc ttc ctg agc gct 1074Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala310 315 320 325ctc gag gag tac act aag aag ctc aac acc cag taagcatgca agcttgaatt 1127Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln330 335ccgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgcctgc caccgctgag 1187ctgagcaata actagcataa cccctctgcc accgctgtgg ggcctctaaa cgggtcttga 1247ggggtttttt gctgaaagga ggaactatat ccgat 128244336PRTArtificial SequencepT7 H6UbiFx Apo A-1 plasmid 44Met Gly Ser His His His His His His Gly Ser Gln Ile Phe Val Lys1 5 10 15Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr20 25 30Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro35 40 45Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg50 55 60Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val65 70 75 80Leu Arg Leu Arg Gly Gly Ser Ile Glu Gly Arg Gly Gly Asp Glu Pro85 90 95Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val100 105 110Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly115 120 125Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp130 135 140Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val145 150 155 160Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg165 170 175Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro180 185 190Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr195 200 205Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg210 215 220Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu225 230 235 240Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu245 250 255Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu260 265 270Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys275 280 285Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu290 295 300Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val305 310 315 320Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln325 330 335451285DNAArtificial SequenceT7 H6UbiFx Cys-Apo A-1 plasmid 45gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac gga tca cag atc ttt gtg aag acc ctc act ggc aaa 162His His His His Gly Ser Gln Ile Phe Val Lys Thr Leu Thr Gly Lys10 15 20acc atc acc ctt gag gtc gag ccc agt gac acc att gag aat gtc aaa 210Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys25 30 35gcc aaa att caa gac aag gag ggt atc cca cct gac cag cag cgt ctg 258Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu40 45 50ata ttt gcc ggc aaa cag ctg gaa gat gga cgt act ttg tct gac tac 306Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr55 60 65aat att caa aag gag tct act ctt cat ctt gtg ttg aga ctt cgt ggt 354Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly70 75 80 85gga tcc atc gag ggt agg ggt gga tgt gat gaa ccc ccc cag agc ccc 402Gly Ser Ile Glu Gly Arg Gly Gly Cys Asp Glu Pro Pro Gln Ser Pro90 95 100tgg gat cga gtg aag gac ctg gcc act gtg tac gtg gat gtg ctc aaa 450Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys105 110 115gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc gcc ttg gga 498Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly120 125 130aaa cag cta aac cta aag ctc ctt gac aac tgg gac agc gtg acc tcc 546Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser135 140 145acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc cag gag ttc 594Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe150 155 160 165tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag gag atg agc 642Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser170 175 180aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac ctg gac gac 690Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp185 190 195ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc cag aag gtg 738Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val200 205 210gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag aag ctg cac 786Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His215 220 225gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg cgc gac cgc 834Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg230 235 240 245gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc ccc tac agc 882Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser250 255 260gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct ctc aag gag 930Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu265 270 275aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc acc gag cat 978Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His280 285 290ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag gac ctc cgc 1026Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg295 300 305caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc ttc ctg agc 1074Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser310 315 320 325gct ctc gag gag tac act aag aag ctc aac acc cag taagcatgca 1120Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln330 335agcttgaatt ccgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgcctgc 1180caccgctgag ctgagcaata actagcataa cccctctgcc accgctgtgg ggcctctaaa 1240cgggtcttga ggggtttttt gctgaaagga ggaactatat ccgat 128546337PRTArtificial SequenceT7 H6UbiFx Cys-Apo A-1 plasmid 46Met Gly Ser His His His His His His Gly Ser Gln Ile Phe Val Lys1 5 10 15Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr20 25 30Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro35 40 45Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg50 55 60Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val65 70 75 80Leu Arg Leu Arg Gly Gly Ser Ile Glu Gly Arg Gly Gly Cys Asp Glu85 90 95Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr100 105 110Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu115 120 125Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp130 135 140Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro145 150 155 160Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu165 170 175Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln180 185 190Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu195 200 205Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala210 215 220Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu225 230 235 240Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His245 250 255Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu260 265 270Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala275 280 285Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala290 295 300Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys305 310 315 320Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr325 330 335Gln471217DNAArtificial SequencepT7 H6 Trip-A-Apo A-1 - AmpR plasmid 47gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac gga tcg atc cag ggt aga tct cct ggt acc gag cca 162His His His His Gly Ser Ile Gln Gly Arg Ser Pro Gly Thr Glu Pro10 15 20cca acc cag aag ccc aag aag att gta aat gcc aag aaa gat gtt gtg 210Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys Lys Asp Val Val25 30 35aac aca aag atg ttt gag gag ctc aag agc cgt ctg gac acc ctg gcc 258Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu Asp Thr Leu Ala40 45 50cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg cag acg gtc tcc 306Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu Gln Thr Val Ser55 60 65ctg aag gga tcc gat gaa ccc ccc cag agc ccc tgg gat cga gtg aag 354Leu Lys Gly Ser Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys70 75 80 85gac ctg gcc act gtg tac gtg gat gtg ctc aaa gac agc ggc aga gac 402Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp90 95 100tat gtg tcc cag ttt gaa ggc tcc gcc ttg gga aaa cag cta aac cta 450Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu105 110 115aag ctc ctt gac aac tgg gac agc gtg acc tcc acc ttc agc aag ctg 498Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu120 125 130cgc gaa cag ctc ggc cct gtg acc cag gag ttc tgg gat aac ctg gaa 546Arg

Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu135 140 145aag gag aca gag ggc ctg agg cag gag atg agc aag gat ctg gag gag 594Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu150 155 160 165gtg aag gcc aag gtg cag ccc tac ctg gac gac ttc cag aag aag tgg 642Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp170 175 180cag gag gag atg gag ctc tac cgc cag aag gtg gag ccg ctg cgc gca 690Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala185 190 195gag ctc caa gag ggc gcg cgc cag aag ctg cac gag ctg caa gag aag 738Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys200 205 210ctg agc cca ctg ggc gag gag atg cgc gac cgc gcg cgc gcc cat gtg 786Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val215 220 225gac gcg ctg cgc acg cat ctg gcc ccc tac agc gac gag ctg cgc cag 834Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln230 235 240 245cgc ttg gcc gcg cgc ctt gag gct ctc aag gag aac ggc ggc gcc aga 882Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg250 255 260ctg gcc gag tac cac gcc aag gcc acc gag cat ctg agc acg ctc agc 930Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser265 270 275gag aag gcc aag ccc gcg ctc gag gac ctc cgc caa ggc ctg ctg ccc 978Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro280 285 290gtg ctg gag agc ttc aag gtc agc ttc ctg agc gct ctc gag gag tac 1026Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr295 300 305act aag aag ctc aac acc cag taataagctt gaattccgat ccggctgcta 1077Thr Lys Lys Leu Asn Thr Gln310 315acaaagcccg aaaggaagct gagttggctg cctgccaccg ctgagctgag caataactag 1137cataacccct ctgccaccgc tgtggggcct ctaaacgggt cttgaggggt tttttgctga 1197aaggaggaac tatatccgat 121748316PRTArtificial SequencepT7 H6 Trip-A-Apo A-1 - AmpR plasmid 48Met Gly Ser His His His His His His Gly Ser Ile Gln Gly Arg Ser1 5 10 15Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala20 25 30Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg35 40 45Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala50 55 60Leu Gln Thr Val Ser Leu Lys Gly Ser Asp Glu Pro Pro Gln Ser Pro65 70 75 80Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys85 90 95Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly100 105 110Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser115 120 125Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe130 135 140Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser145 150 155 160Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp165 170 175Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val180 185 190Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His195 200 205Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg210 215 220Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser225 230 235 240Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu245 250 255Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His260 265 270Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg275 280 285Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser290 295 300Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln305 310 315491088DNAArtificial SequencepT7H6 Trip-A-Apo A-1-del 43 - AmpR plasmid 49gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac gga tcg atc cag ggt aga tct cct ggt acc gag cca 162His His His His Gly Ser Ile Gln Gly Arg Ser Pro Gly Thr Glu Pro10 15 20cca acc cag aag ccc aag aag att gta aat gcc aag aaa gat gtt gtg 210Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys Lys Asp Val Val25 30 35aac aca aag atg ttt gag gag ctc aag agc cgt ctg gac acc ctg gcc 258Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu Asp Thr Leu Ala40 45 50cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg cag acg gtc tcc 306Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu Gln Thr Val Ser55 60 65ctg aag gga tcc cta aag ctc ctt gac aac tgg gac agc gtg acc tcc 354Leu Lys Gly Ser Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser70 75 80 85acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc cag gag ttc 402Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe90 95 100tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag gag atg agc 450Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser105 110 115aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac ctg gac gac 498Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp120 125 130ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc cag aag gtg 546Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val135 140 145gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag aag ctg cac 594Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His150 155 160 165gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg cgc gac cgc 642Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg170 175 180gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc ccc tac agc 690Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser185 190 195gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct ctc aag gag 738Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu200 205 210aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc acc gag cat 786Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His215 220 225ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag gac ctc cgc 834Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg230 235 240 245caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc ttc ctg agc 882Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser250 255 260gct ctc gag gag tac act aag aag ctc aac acc cag taataagctt 928Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln265 270gaattccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg cctgccaccg 988ctgagctgag caataactag cataacccct ctgccaccgc tgtggggcct ctaaacgggt 1048cttgaggggt tttttgctga aaggaggaac tatatccgat 108850273PRTArtificial SequencepT7H6 Trip-A-Apo A-1-del 43 - AmpR plasmid 50Met Gly Ser His His His His His His Gly Ser Ile Gln Gly Arg Ser1 5 10 15Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala20 25 30Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg35 40 45Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala50 55 60Leu Gln Thr Val Ser Leu Lys Gly Ser Leu Lys Leu Leu Asp Asn Trp65 70 75 80Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro85 90 95Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu100 105 110Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln115 120 125Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu130 135 140Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala145 150 155 160Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu165 170 175Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His180 185 190Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu195 200 205Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala210 215 220Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala225 230 235 240Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys245 250 255Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr260 265 270Gln511057DNAArtificial SequencepT7 H6 Fx Cys-Apo A1 plasmid 51gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac gga tcc atc gag ggt agg ggt gga tgt gat gaa ccc 162His His His His Gly Ser Ile Glu Gly Arg Gly Gly Cys Asp Glu Pro10 15 20ccc cag agc ccc tgg gat cga gtg aag gac ctg gcc act gtg tac gtg 210Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val25 30 35gat gtg ctc aaa gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc 258Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly40 45 50tcc gcc ttg gga aaa cag cta aac cta aag ctc ctt gac aac tgg gac 306Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp55 60 65agc gtg acc tcc acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg 354Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val70 75 80 85acc cag gag ttc tgg gat aac ctg gaa aag gag aca gag ggc ctg agg 402Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg90 95 100cag gag atg agc aag gat ctg gag gag gtg aag gcc aag gtg cag ccc 450Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro105 110 115tac ctg gac gac ttc cag aag aag tgg cag gag gag atg gag ctc tac 498Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr120 125 130cgc cag aag gtg gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc 546Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg135 140 145cag aag ctg cac gag ctg caa gag aag ctg agc cca ctg ggc gag gag 594Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu150 155 160 165atg cgc gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg 642Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu170 175 180gcc ccc tac agc gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag 690Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu185 190 195gct ctc aag gag aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag 738Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys200 205 210gcc acc gag cat ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc 786Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu215 220 225gag gac ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc 834Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val230 235 240 245agc ttc ctg agc gct ctc gag gag tac act aag aag ctc aac acc cag 882Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln250 255 260taagcatgca agcttgaatt ccgatccggc tgctaacaaa gcccgaaagg aagctgagtt 942ggctgcctgc caccgctgag ctgagcaata actagcataa cccctctgcc accgctgtgg 1002ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccgat 105752261PRTArtificial SequencepT7 H6 Fx Cys-Apo A1 plasmid 52Met Gly Ser His His His His His His Gly Ser Ile Glu Gly Arg Gly1 5 10 15Gly Cys Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu20 25 30Ala Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val35 40 45Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu50 55 60Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu65 70 75 80Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu85 90 95Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys100 105 110Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu115 120 125Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu130 135 140Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser145 150 155 160Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala165 170 175Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu180 185 190Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala195 200 205Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys210 215 220Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu225 230 235 240Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys245 250 255Lys Leu Asn Thr Gln260531217DNAArtificial SequencepT7H6 Trip-A-Apo A1 K9A K15A - AmpR plasmid 53gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac gga tcg atc cag ggt aga tct cct ggt acc gag cca 162His His His His Gly Ser Ile Gln Gly Arg Ser Pro Gly Thr Glu Pro10 15 20cca acc cag aag ccc aag gcg att gta aat gcc aag gca gat gtt gtg 210Pro Thr Gln Lys Pro Lys Ala Ile Val Asn Ala Lys Ala Asp Val Val25 30 35aac aca aag atg ttt gag gag ctc aag agc cgt ctg gac acc ctg gcc 258Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu Asp Thr Leu Ala40 45 50cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg cag acg gtc tcc 306Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu Gln Thr Val Ser55 60 65ctg aag gga tcc gat gaa ccc ccc cag agc ccc tgg gat cga gtg aag 354Leu Lys Gly Ser Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys70 75 80 85gac ctg gcc act gtg tac gtg gat gtg ctc aaa gac agc ggc aga gac 402Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp90 95 100tat gtg tcc cag ttt gaa ggc tcc gcc ttg gga aaa cag cta aac cta 450Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu105 110 115aag ctc ctt gac aac tgg gac agc gtg acc tcc acc ttc agc aag ctg 498Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu120 125 130cgc gaa cag ctc ggc cct gtg acc cag gag ttc tgg gat aac ctg gaa 546Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu135 140 145aag gag aca gag ggc ctg agg cag gag atg agc aag gat ctg gag gag 594Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu150 155 160 165gtg aag gcc aag gtg cag ccc tac ctg gac gac ttc cag aag aag tgg 642Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp170 175 180cag gag gag atg gag ctc tac cgc cag aag gtg gag ccg ctg cgc gca 690Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala185 190 195gag ctc caa gag ggc gcg cgc cag aag ctg cac gag ctg caa gag aag 738Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys200 205 210ctg agc cca ctg ggc gag gag atg cgc gac cgc gcg cgc gcc cat gtg 786Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val215 220 225gac gcg ctg cgc acg cat ctg gcc ccc tac agc gac gag ctg cgc cag 834Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln230 235 240 245cgc ttg gcc gcg cgc ctt gag gct ctc aag gag aac ggc ggc gcc aga

882Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg250 255 260ctg gcc gag tac cac gcc aag gcc acc gag cat ctg agc acg ctc agc 930Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser265 270 275gag aag gcc aag ccc gcg ctc gag gac ctc cgc caa ggc ctg ctg ccc 978Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro280 285 290gtg ctg gag agc ttc aag gtc agc ttc ctg agc gct ctc gag gag tac 1026Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr295 300 305act aag aag ctc aac acc cag taataagctt gaattccgat ccggctgcta 1077Thr Lys Lys Leu Asn Thr Gln310 315acaaagcccg aaaggaagct gagttggctg cctgccaccg ctgagctgag caataactag 1137cataacccct ctgccaccgc tgtggggcct ctaaacgggt cttgaggggt tttttgctga 1197aaggaggaac tatatccgat 121754316PRTArtificial SequencepT7H6 Trip-A-Apo A1 K9A K15A - AmpR plasmid 54Met Gly Ser His His His His His His Gly Ser Ile Gln Gly Arg Ser1 5 10 15Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala Ile Val Asn Ala20 25 30Lys Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg35 40 45Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala50 55 60Leu Gln Thr Val Ser Leu Lys Gly Ser Asp Glu Pro Pro Gln Ser Pro65 70 75 80Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys85 90 95Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly100 105 110Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser115 120 125Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe130 135 140Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser145 150 155 160Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp165 170 175Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val180 185 190Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His195 200 205Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg210 215 220Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser225 230 235 240Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu245 250 255Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His260 265 270Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg275 280 285Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser290 295 300Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln305 310 315551238DNAArtificial SequencepT7H6 Trip-A-Fn-Apo A1 - AmpR plasmid 55gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac ggt agt ggt agt gga tca atc cag ggt aga tct cct 162His His His His Gly Ser Gly Ser Gly Ser Ile Gln Gly Arg Ser Pro10 15 20ggt acc gag cca cca acc cag aag ccc aag aag att gta aat gcc aag 210Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys25 30 35aaa gat gtt gtg aac aca aag atg ttt gag gag ctc aag agc cgt ctg 258Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu40 45 50gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg 306Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu55 60 65cag acg gtc tcc ctg aag gga tcc tcg ggt cat gat gaa ccc ccc cag 354Gln Thr Val Ser Leu Lys Gly Ser Ser Gly His Asp Glu Pro Pro Gln70 75 80 85agc ccc tgg gat cga gtg aag gac ctg gcc act gtg tac gtg gat gtg 402Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val90 95 100ctc aaa gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc gcc 450Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala105 110 115ttg gga aaa cag cta aac cta aag ctc ctt gac aac tgg gac agc gtg 498Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val120 125 130acc tcc acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc cag 546Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln135 140 145gag ttc tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag gag 594Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu150 155 160 165atg agc aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac ctg 642Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu170 175 180gac gac ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc cag 690Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln185 190 195aag gtg gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag aag 738Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys200 205 210ctg cac gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg cgc 786Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg215 220 225gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc ccc 834Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro230 235 240 245tac agc gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct ctc 882Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu250 255 260aag gag aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc acc 930Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr265 270 275gag cat ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag gac 978Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp280 285 290ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc ttc 1026Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe295 300 305ctg agc gct ctc gag gag tac act aag aag ctc aac acc cag 1068Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln310 315 320taataagctt gaattccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 1128cctgccaccg ctgagctgag caataactag cataacccct ctgccaccgc tgtggggcct 1188ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgat 123856323PRTArtificial SequencepT7H6 Trip-A-Fn-Apo A1 - AmpR plasmid 56Met Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile1 5 10 15Gln Gly Arg Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys20 25 30Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu35 40 45Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys50 55 60Glu Gln Gln Ala Leu Gln Thr Val Ser Leu Lys Gly Ser Ser Gly His65 70 75 80Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr85 90 95Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln100 105 110Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp115 120 125Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu130 135 140Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu145 150 155 160Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys165 170 175Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met180 185 190Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu195 200 205Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu210 215 220Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg225 230 235 240Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala245 250 255Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr260 265 270His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys275 280 285Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser290 295 300Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu305 310 315 320Asn Thr Gln571238DNAArtificial SequencepT7H6 Trip-A-Fn-Apo A1-final - AmpR plasmid 57gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac ggt agt ggt agt gga tca atc cag ggt aga tct cct 162His His His His Gly Ser Gly Ser Gly Ser Ile Gln Gly Arg Ser Pro10 15 20ggt acc gag cca cca acc cag aag ccc aag aag att gta aat gcc aag 210Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys25 30 35aaa gat gtt gtg aac aca aag atg ttt gag gag ctc aag agc cgt ctg 258Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu40 45 50gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg 306Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu55 60 65cag acg gtc tcc ctg aag gga acc tcg ggt cag gat gaa ccc ccc cag 354Gln Thr Val Ser Leu Lys Gly Thr Ser Gly Gln Asp Glu Pro Pro Gln70 75 80 85agc ccc tgg gat cga gtg aag gac ctg gcc act gtg tac gtg gat gtg 402Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val90 95 100ctc aaa gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc gcc 450Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala105 110 115ttg gga aaa cag cta aac cta aag ctc ctt gac aac tgg gac agc gtg 498Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val120 125 130acc tcc acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc cag 546Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln135 140 145gag ttc tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag gag 594Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu150 155 160 165atg agc aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac ctg 642Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu170 175 180gac gac ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc cag 690Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln185 190 195aag gtg gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag aag 738Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys200 205 210ctg cac gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg cgc 786Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg215 220 225gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc ccc 834Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro230 235 240 245tac agc gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct ctc 882Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu250 255 260aag gag aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc acc 930Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr265 270 275gag cat ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag gac 978Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp280 285 290ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc ttc 1026Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe295 300 305ctg agc gct ctc gag gag tac act aag aag ctc aac acc cag 1068Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln310 315 320taataagctt gaattccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 1128cctgccaccg ctgagctgag caataactag cataacccct ctgccaccgc tgtggggcct 1188ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgat 123858323PRTArtificial SequencepT7H6 Trip-A-Fn-Apo A1-final - AmpR plasmid 58Met Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile1 5 10 15Gln Gly Arg Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys20 25 30Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu35 40 45Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys50 55 60Glu Gln Gln Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Ser Gly Gln65 70 75 80Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr85 90 95Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln100 105 110Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp115 120 125Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu130 135 140Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu145 150 155 160Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys165 170 175Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met180 185 190Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu195 200 205Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu210 215 220Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg225 230 235 240Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala245 250 255Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr260 265 270His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys275 280 285Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser290 295 300Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu305 310 315 320Asn Thr Gln591238DNAArtificial SequencepT7H6 Trip-A-Fn-Apo A1 final K9AK15A - AmpR plasmid 59gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac ggt agt ggt agt gga tca atc cag ggt aga tct cct 162His His His His Gly Ser Gly Ser Gly Ser Ile Gln Gly Arg Ser Pro10 15 20ggt acc gag cca cca acc cag aag ccc aag gcg att gta aat gcc aag 210Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala Ile Val Asn Ala Lys25 30 35gca gat gtt gtg aac aca aag atg ttt gag gag ctc aag agc cgt ctg 258Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu40 45 50gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg 306Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu55 60 65cag acg gtc tcc ctg aag gga acc tcg ggt cag gat gaa ccc ccc cag 354Gln Thr Val Ser Leu Lys Gly Thr Ser Gly Gln Asp Glu Pro Pro Gln70 75 80 85agc ccc tgg gat cga gtg aag gac ctg gcc act gtg tac gtg gat gtg 402Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val90 95 100ctc aaa gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc gcc 450Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala105 110 115ttg gga aaa cag cta aac cta aag ctc ctt gac aac tgg gac agc gtg 498Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val120 125 130acc tcc acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc cag 546Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln135 140 145gag ttc tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag gag 594Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu150 155 160 165atg agc aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac ctg 642Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu170 175

180gac gac ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc cag 690Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln185 190 195aag gtg gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag aag 738Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys200 205 210ctg cac gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg cgc 786Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg215 220 225gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc ccc 834Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro230 235 240 245tac agc gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct ctc 882Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu250 255 260aag gag aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc acc 930Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr265 270 275gag cat ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag gac 978Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp280 285 290ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc ttc 1026Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe295 300 305ctg agc gct ctc gag gag tac act aag aag ctc aac acc cag 1068Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln310 315 320taataagctt gaattccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 1128cctgccaccg ctgagctgag caataactag cataacccct ctgccaccgc tgtggggcct 1188ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgat 123860323PRTArtificial SequencepT7H6 Trip-A-Fn-Apo A1 final K9AK15A - AmpR plasmid 60Met Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile1 5 10 15Gln Gly Arg Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala20 25 30Ile Val Asn Ala Lys Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu35 40 45Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys50 55 60Glu Gln Gln Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Ser Gly Gln65 70 75 80Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr85 90 95Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln100 105 110Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp115 120 125Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu130 135 140Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu145 150 155 160Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys165 170 175Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met180 185 190Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu195 200 205Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu210 215 220Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg225 230 235 240Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala245 250 255Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr260 265 270His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys275 280 285Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser290 295 300Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu305 310 315 320Asn Thr Gln611241DNAArtificial SequencepT7H6 (GS)3 Trip-A-Tn-Apo A1 AmpR plasmid 61gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac ggt agt ggt agt gga tca atc cag ggt aga tct cct 162His His His His Gly Ser Gly Ser Gly Ser Ile Gln Gly Arg Ser Pro10 15 20ggt acc gag cca cca acc cag aag ccc aag aag att gta aat gcc aag 210Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys25 30 35aaa gat gtt gtg aac aca aag atg ttt gag gag ctc aag agc cgt ctg 258Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu40 45 50gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg 306Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu55 60 65cag acg gtc tcc ctg aag gga tcc aag gtg cac atg aag gaa ccc ccc 354Gln Thr Val Ser Leu Lys Gly Ser Lys Val His Met Lys Glu Pro Pro70 75 80 85cag agc ccc tgg gat cga gtg aag gac ctg gcc act gtg tac gtg gat 402Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp90 95 100gtg ctc aaa gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc 450Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser105 110 115gcc ttg gga aaa cag cta aac cta aag ctc ctt gac aac tgg gac agc 498Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser120 125 130gtg acc tcc acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc 546Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr135 140 145cag gag ttc tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag 594Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln150 155 160 165gag atg agc aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac 642Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr170 175 180ctg gac gac ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc 690Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg185 190 195cag aag gtg gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag 738Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln200 205 210aag ctg cac gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg 786Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met215 220 225cgc gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc 834Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala230 235 240 245ccc tac agc gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct 882Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala250 255 260ctc aag gag aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc 930Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala265 270 275acc gag cat ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag 978Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu280 285 290gac ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc 1026Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser295 300 305ttc ctg agc gct ctc gag gag tac act aag aag ctc aac acc cag 1071Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln310 315 320taataagctt gaattccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 1131cctgccaccg ctgagctgag caataactag cataacccct ctgccaccgc tgtggggcct 1191ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgat 124162324PRTArtificial SequencepT7H6 (GS)3 Trip-A-Tn-Apo A1 AmpR plasmid 62Met Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile1 5 10 15Gln Gly Arg Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys20 25 30Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu35 40 45Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys50 55 60Glu Gln Gln Ala Leu Gln Thr Val Ser Leu Lys Gly Ser Lys Val His65 70 75 80Met Lys Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala85 90 95Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser100 105 110Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu115 120 125Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln130 135 140Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr145 150 155 160Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala165 170 175Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu180 185 190Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln195 200 205Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro210 215 220Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu225 230 235 240Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala245 250 255Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu260 265 270Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala275 280 285Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu290 295 300Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys305 310 315 320Leu Asn Thr Gln631241DNAArtificial SequencepT7H6 Trip-A-Tn-Apo A1-final - AmpR plasmid 63gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac ggt agt ggt agt gga tca atc cag ggt aga tct cct 162His His His His Gly Ser Gly Ser Gly Ser Ile Gln Gly Arg Ser Pro10 15 20ggt acc gag cca cca acc cag aag ccc aag aag att gta aat gcc aag 210Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys25 30 35aaa gat gtt gtg aac aca aag atg ttt gag gag ctc aag agc cgt ctg 258Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu40 45 50gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg 306Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu55 60 65cag acg gtc tcc ctg aag gga acc aag gtg cac atg aag gaa ccc ccc 354Gln Thr Val Ser Leu Lys Gly Thr Lys Val His Met Lys Glu Pro Pro70 75 80 85cag agc ccc tgg gat cga gtg aag gac ctg gcc act gtg tac gtg gat 402Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp90 95 100gtg ctc aaa gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc 450Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser105 110 115gcc ttg gga aaa cag cta aac cta aag ctc ctt gac aac tgg gac agc 498Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser120 125 130gtg acc tcc acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc 546Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr135 140 145cag gag ttc tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag 594Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln150 155 160 165gag atg agc aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac 642Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr170 175 180ctg gac gac ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc 690Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg185 190 195cag aag gtg gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag 738Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln200 205 210aag ctg cac gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg 786Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met215 220 225cgc gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc 834Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala230 235 240 245ccc tac agc gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct 882Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala250 255 260ctc aag gag aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc 930Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala265 270 275acc gag cat ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag 978Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu280 285 290gac ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc 1026Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser295 300 305ttc ctg agc gct ctc gag gag tac act aag aag ctc aac acc cag 1071Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln310 315 320taataagctt gaattccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 1131cctgccaccg ctgagctgag caataactag cataacccct ctgccaccgc tgtggggcct 1191ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgat 124164324PRTArtificial SequencepT7H6 Trip-A-Tn-Apo A1-final - AmpR plasmid 64Met Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile1 5 10 15Gln Gly Arg Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys20 25 30Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu35 40 45Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys50 55 60Glu Gln Gln Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Lys Val His65 70 75 80Met Lys Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala85 90 95Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser100 105 110Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu115 120 125Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln130 135 140Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr145 150 155 160Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala165 170 175Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu180 185 190Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln195 200 205Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro210 215 220Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu225 230 235 240Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala245 250 255Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu260 265 270Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala275 280 285Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu290 295 300Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys305 310 315 320Leu Asn Thr Gln651241DNAArtificial SequencepT7H6 Trip-A-Tn-Apo A1 final K9AK15A - AmpR plasmid 65gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac ggt agt ggt agt gga tca atc cag ggt aga tct cct 162His His His His Gly Ser Gly Ser Gly Ser Ile Gln Gly Arg Ser Pro10 15 20ggt acc gag cca cca acc cag aag ccc aag gcg att gta aat gcc aag 210Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala Ile Val Asn Ala Lys25 30 35gca gat gtt gtg aac aca aag atg ttt gag gag ctc aag agc cgt ctg 258Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu40 45 50gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag cag cag gcc ctg 306Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu55 60 65cag acg gtc tcc ctg aag gga acc aag gtg cac atg aag gaa ccc ccc 354Gln Thr Val Ser Leu Lys Gly Thr Lys Val His Met Lys Glu Pro Pro70 75 80 85cag agc ccc tgg gat cga gtg aag gac ctg gcc act gtg tac gtg gat 402Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala

Thr Val Tyr Val Asp90 95 100gtg ctc aaa gac agc ggc aga gac tat gtg tcc cag ttt gaa ggc tcc 450Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser105 110 115gcc ttg gga aaa cag cta aac cta aag ctc ctt gac aac tgg gac agc 498Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser120 125 130gtg acc tcc acc ttc agc aag ctg cgc gaa cag ctc ggc cct gtg acc 546Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr135 140 145cag gag ttc tgg gat aac ctg gaa aag gag aca gag ggc ctg agg cag 594Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln150 155 160 165gag atg agc aag gat ctg gag gag gtg aag gcc aag gtg cag ccc tac 642Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr170 175 180ctg gac gac ttc cag aag aag tgg cag gag gag atg gag ctc tac cgc 690Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg185 190 195cag aag gtg gag ccg ctg cgc gca gag ctc caa gag ggc gcg cgc cag 738Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln200 205 210aag ctg cac gag ctg caa gag aag ctg agc cca ctg ggc gag gag atg 786Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met215 220 225cgc gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc acg cat ctg gcc 834Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala230 235 240 245ccc tac agc gac gag ctg cgc cag cgc ttg gcc gcg cgc ctt gag gct 882Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala250 255 260ctc aag gag aac ggc ggc gcc aga ctg gcc gag tac cac gcc aag gcc 930Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala265 270 275acc gag cat ctg agc acg ctc agc gag aag gcc aag ccc gcg ctc gag 978Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu280 285 290gac ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc ttc aag gtc agc 1026Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser295 300 305ttc ctg agc gct ctc gag gag tac act aag aag ctc aac acc cag 1071Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln310 315 320taataagctt gaattccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 1131cctgccaccg ctgagctgag caataactag cataacccct ctgccaccgc tgtggggcct 1191ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgat 124166324PRTArtificial SequencepT7H6 Trip-A-Tn-Apo A1 final K9AK15A - AmpR plasmid 66Met Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile1 5 10 15Gln Gly Arg Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Ala20 25 30Ile Val Asn Ala Lys Ala Asp Val Val Asn Thr Lys Met Phe Glu Glu35 40 45Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys50 55 60Glu Gln Gln Ala Leu Gln Thr Val Ser Leu Lys Gly Thr Lys Val His65 70 75 80Met Lys Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala85 90 95Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser100 105 110Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu115 120 125Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln130 135 140Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr145 150 155 160Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala165 170 175Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu180 185 190Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln195 200 205Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro210 215 220Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu225 230 235 240Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala245 250 255Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu260 265 270Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala275 280 285Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu290 295 300Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys305 310 315 320Leu Asn Thr Gln671301DNAArtificial SequencepT7H6 Hp-alpha-Apo A1 - AmpR plasmid 67gatctcgatc ccgcgaaatt aatacgatac actataggga gaccacaacg gtttccctct 60agaaataatt ttgtttaact ttaagaagga gatatacat atg gga tcg cat cac 114Met Gly Ser His His1 5cat cac cat cac gga tcg atc cag ggt aga ggt gtg gac tca ggc aat 162His His His His Gly Ser Ile Gln Gly Arg Gly Val Asp Ser Gly Asn10 15 20gat gtc acg gat atc gca gat gac ggc tgc ccg aag ccc ccc gag att 210Asp Val Thr Asp Ile Ala Asp Asp Gly Cys Pro Lys Pro Pro Glu Ile25 30 35gca cat ggc tat gtg gag cac tcg gtt cgc tac cag tgt aag aac tac 258Ala His Gly Tyr Val Glu His Ser Val Arg Tyr Gln Cys Lys Asn Tyr40 45 50tac aaa ctg cgc aca gaa gga gat gga gta tac acc tta aac aat gag 306Tyr Lys Leu Arg Thr Glu Gly Asp Gly Val Tyr Thr Leu Asn Asn Glu55 60 65aag cag tgg ata aat aag gct gtt gga gat aaa ctt cct gaa tgt gaa 354Lys Gln Trp Ile Asn Lys Ala Val Gly Asp Lys Leu Pro Glu Cys Glu70 75 80 85gca gta gct ggg aag ccc aag aat ccg gca aac cca gtg cag aga tcc 402Ala Val Ala Gly Lys Pro Lys Asn Pro Ala Asn Pro Val Gln Arg Ser90 95 100gat gaa ccc ccc cag agc ccc tgg gat cga gtg aag gac ctg gcc act 450Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr105 110 115gtg tac gtg gat gtg ctc aaa gac agc ggc aga gac tat gtg tcc cag 498Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln120 125 130ttt gaa ggc tcc gcc ttg gga aaa cag cta aac cta aag ctc ctt gac 546Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp135 140 145aac tgg gac agc gtg acc tcc acc ttc agc aag ctg cgc gaa cag ctc 594Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu150 155 160 165ggc cct gtg acc cag gag ttc tgg gat aac ctg gaa aag gag aca gag 642Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu170 175 180ggc ctg agg cag gag atg agc aag gat ctg gag gag gtg aag gcc aag 690Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys185 190 195gtg cag ccc tac ctg gac gac ttc cag aag aag tgg cag gag gag atg 738Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met200 205 210gag ctc tac cgc cag aag gtg gag ccg ctg cgc gca gag ctc caa gag 786Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu215 220 225ggc gcg cgc cag aag ctg cac gag ctg caa gag aag ctg agc cca ctg 834Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu230 235 240 245ggc gag gag atg cgc gac cgc gcg cgc gcc cat gtg gac gcg ctg cgc 882Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg250 255 260acg cat ctg gcc ccc tac agc gac gag ctg cgc cag cgc ttg gcc gcg 930Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala265 270 275cgc ctt gag gct ctc aag gag aac ggc ggc gcc aga ctg gcc gag tac 978Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr280 285 290cac gcc aag gcc acc gag cat ctg agc acg ctc agc gag aag gcc aag 1026His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys295 300 305ccc gcg ctc gag gac ctc cgc caa ggc ctg ctg ccc gtg ctg gag agc 1074Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser310 315 320 325ttc aag gtc agc ttc ctg agc gct ctc gag gag tac act aag aag ctc 1122Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu330 335 340aac acc cag taataagctt gaattccgat ccggctgcta acaaagcccg 1171Asn Thr Glnaaaggaagct gagttggctg cctgccaccg ctgagctgag caataactag cataacccct 1231ctgccaccgc tgtggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac 1291tatatccgat 130168344PRTArtificial SequencepT7H6 Hp-alpha-Apo A1 - AmpR plasmid 68Met Gly Ser His His His His His His Gly Ser Ile Gln Gly Arg Gly1 5 10 15Val Asp Ser Gly Asn Asp Val Thr Asp Ile Ala Asp Asp Gly Cys Pro20 25 30Lys Pro Pro Glu Ile Ala His Gly Tyr Val Glu His Ser Val Arg Tyr35 40 45Gln Cys Lys Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly Val Tyr50 55 60Thr Leu Asn Asn Glu Lys Gln Trp Ile Asn Lys Ala Val Gly Asp Lys65 70 75 80Leu Pro Glu Cys Glu Ala Val Ala Gly Lys Pro Lys Asn Pro Ala Asn85 90 95Pro Val Gln Arg Ser Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val100 105 110Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg115 120 125Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn130 135 140Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys145 150 155 160Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu165 170 175Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu180 185 190Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys195 200 205Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg210 215 220Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu225 230 235 240Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His245 250 255Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg260 265 270Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala275 280 285Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu290 295 300Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu305 310 315 320Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu325 330 335Tyr Thr Lys Lys Leu Asn Thr Gln340697PRTArtificial Sequencetetranectin (53-59) based linker 69Gly Thr Lys Val His Met Lys1 57013PRTArtificial Sequencefibronectin (2037-2049) based linker 70Pro Gly Thr Ser Gly Gln Gln Pro Ser Val Gly Gln Gln1 5 107110PRTArtificial Sequencemurine IgG3 upper hinge region-based linker 71Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser1 5 107214PRTArtificial Sequenceflexible linker (Muller, 2000) 72Ser Gly Gly Thr Ser Gly Ser Thr Ser Gly Thr Gly Ser Thr1 5 107315PRTArtificial Sequenceflexible linker (Muller, 2000) 73Ala Gly Ser Ser Thr Gly Ser Ser Thr Gly Pro Gly Ser Thr Thr1 5 10 15747PRTArtificial Sequenceflexible linker (Muller, 2000) 74Gly Gly Ser Gly Gly Ala Pro1 57545DNAArtificial SequenceUbi-A-I primer #1 75cacggatcca tcgagggtag gggtggagat gaaccccccc agagc 457635DNAArtificial SequenceUbi-A-I primer #2 76tccaagctta ttactgggtg ttgagcttct tagtg 357730DNAArtificial SequenceTrip-A-A-I primer #1 77aagggatccg atgaaccccc ccagagcccc 307835DNAArtificial SequenceTrip-A-A-I primer #2 78tccaagctta ttactgggtg ttgagcttct tagtg 357931DNAArtificial SequenceTrip-A-I-del43 primer #1 79aggggatccc taaagctcct tgacaactgg g 318035DNAArtificial SequenceTrip-A-I-del43 primer #2 80tccaagctta ttactgggtg ttgagcttct tagtg 358143DNAArtificial SequenceUbi-Cys-A-I primer #1 81ggtggatcca tcgagggtag gggtggatgt gatgaacccc ccc 438235DNAArtificial SequenceUbi-Cys-A-I primer #2 82tccaagctta ttactgggtg ttgagcttct tagtg 358339DNAArtificial SequencepT7H6Fx-Trip-A-FN(-2)-AI primer 83cgcggatcct cgggtcagga tgaacccccc cagagcccc 398445DNAArtificial SequencepT7H6Fx-Trip-A-TN-AI-Bam-S primer 84cgcggatcca aggtgcacat gaaggatgaa cccccccaga gcccc 458534DNAArtificial SequencepT7H6Fx-Trip-FN-AI primer #1 85acggtctccc tgaagggaac ctcgggtcag gatg 348637DNAArtificial SequencepT-H6Fx-Trip-TN-AI primer 86acggtctccc tgaagggaac caaggtgcac atgaagg 378733DNAArtificial Sequenceprimer #1 to mutate lysine 9 from Trip-A 87ccaacccaga agcccaaggc gaatgtaaat gcc 338833DNAArtificial Sequenceprimer #2 to mutate lysine 9 from Trip-A 88gtgttcacaa catctgcctt ggcatttaca atc 338933DNAArtificial Sequenceprimer #1 to mutate lysine 15 from Trip-A 89ggcatttaca atcgccttgg gcttctgggt tgg 339048DNAArtificial SequenceHP-alpha-A-I cDNA fetal liver library "nonsense" primer 90cacaagcttt ccgctagatc tctgcactgg gttagccgga ttcttggg 489149DNAArtificial SequenceHP-alpha-A-I fetal liver cDNA library "sense" primer 91ggtggatcca tcgagggtag gggtgtggac tcaggcaatg atgtcacgg 49

Claims

1. A method for the prevention and/or treatment of a disease or condition comprising administering to an individual in need thereof, a pharmaceutical composition comprising an apolipoprotein protein construct having the general formula apo-A-X, where apo-A is an apolipoprotein component selected from the group consisting of apolipoprotein A-I, apolipoprotein A-II, and apolipoprotein A-IV, and X is a tetranectin trimerising module.

2. A method according to claim 1, wherein the apolipoprotein protein construct further comprises a spacer peptide between the apo-A component and the tetranectin trimerising module, wherein the spacer peptide comprises at least two amino acids.

3. A method according to claim 2, wherein the spacer peptide is essentially non-immunogenic, and/or is not prone to proteolytic cleavage and/or does not comprise any cystein residues.

4. A method according to claim 2, wherein the three-dimensional structure of the spacer is linear.

5. A method according to claim 2, wherein the spacer peptide comprises an amino acid sequence selected from the group consisting of GTKVHMK (SEQ ID NO: 69), PGTSGQQPSVGQQ (SEQ ID NO: 70), GTSGQ (residues 2-6 of SEQ ID NO: 70), PKPSTPPGSS (SEQ ID NO: 71), SGGTSGSTSGTGST (SEQ ID NO: 72), AGSSTGSSTGPGSTT (SEQ ID NO: 73) and GGSGGAP (SEQ ID NO: 74).

6. A method according to claim 2, wherein the tetranectin trimerising module is linked by a covalent link to the N-terminal or the C-terminal amino acid of apo-A.

7. A method according to claim 1, wherein the tetranectin trimerising module is part of a stable trimeric complex with two other tetranectin trimerising modules.

8. A method according to claim 7, wherein the stable trimeric complex includes a coiled coil structure.

9. A method according to claim 8, wherein the coiled coil structure is a triple alpha helical coiled coil.

10. A method according to claim 9, wherein the stable trimeric complex comprises two tetranectin trimerising modules linked by a spacer moiety, which allows both of the two tetranectin trimerising modules to take part in the complex formation with a third tetranectin trimerising module not being part of the apolipoprotein fusion protein construct.

11. A method according to claim 1, wherein the tetranectin trimerising module is selected from the group consisting of human tetranectin, murine tetranectin or C-type lectin of human, bovine or shark cartilage.

12. A method according to claim 1, wherein the tetranectin trimerising module comprises a sequence having at least 68% identity with the sequence of SEQ ID NO 12 and is capable of forming a stable trimeric complex with other tetranectin trimerising modules.

13. A method according to claim 12, wherein the cystein residue 50 in SEQ ID NO 12 is substituted by a serine residue, a threonine residue, or a methionine residue.

14. A method according to claim 1, wherein the tetranectin trimerisation module has at least 68% sequence identity with the Trip A module (SEQ ID NO 13) and is capable of forming a stable trimeric complex with other tetranectin trimerising modules.

15. A method according to claim 7, wherein the stable trimeric complex has a half-life at least 2 times the half-life of native apolipoprotein A-I, A-II or A-IV.

16. A method according to claim 7, wherein the stable trimeric complex has a half-life at least 3 times the half-life of native apolipoprotein A-I, A-II or A-IV.

17. A method according to claim 7, wherein the stable trimeric complex has a half-life at least 4 times the half-life of native apolipoprotein A-I, A-II or A-IV.

18. A method according to claim 7, wherein the stable trimeric complex has a half-life at least 10 times the half-life of native apolipoprotein A-I, A-II or A-IV.

19. A method according to claim 7, wherein said stable trimeric complex is capable of binding to a receptor or protein selected from the group consisting of cubilin, megalin, Scavenger receptor class B, type 1 (SR-B1), ATP-binding cassette 1 (ABC1), Lecithin:cholesterol acyltransferase (LCAT), Cholesteryl-ester transfer protein (CETP), and Phospolipid transfer protein (PLTP).

20. A method according to claim 19, wherein the trimeric complex comprises an amino acid sequence having at least 70% sequence identity to one of the sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO C, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11.

21. A method according to claim 7, wherein the composition further comprises pharmaceutical acceptable excipients, adjuvants, or additives.

22. A method according to claim 1, wherein the apolipoprotein A-I is human apolipoprotein A-I.

23. A method according to claim 1, wherein the apolipoprotein A-I is a fragment of human apolipoprotein A-I, wherein said fragment substantially retains the lipid binding function of human apolipoprotein A-I.

24. A method according to claim 23, wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 100-186 of human apolipoprotein A-I.

25. A method according to claim 23, wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 25-267 of human apolipoprotein A-I (SEQ ID NO 1).

26. A method according to claim 23, wherein the fragment of human apolipoprotein A-I is amino acids 68-267 from human apolipoprotein A-I.

27. A method according to claim 1, wherein the individual has a condition related to a disorder selected from cholesterol disorders, phospholipid disorders, triacylglyceride disorders, LDL disorders and HDL disorders.

28. A method according to claim 1, wherein the disease is an arteriosclerotic disease.

29. A method according to claim 1, wherein the disease is a cardiovascular disease.

30. A method according to claim 1, wherein the disease is angina pectoris.

31. A method according to claim 1, wherein the disease is myocardial infarction.

32. A method according to claim 1, wherein the disease is plaque angina pectoris.

33. A method according to claim 1, wherein the disease is unstable angina pectoris.

34. A method according to claim 1, wherein the disease is arterial stenosis.

35. A method according to claim 1, wherein the disease is claudicatio.

36. A method according to claim 1, wherein the disease is carotis stenosis.

37. A method according to claim 1, wherein the disease is cerebral arterial stenosis.

38. A method according to claim 1, wherein the condition is caused by endotoxins.

39. A method according to claim 1, wherein the pharmaceutical composition is administered intravenously, intraarterially, intramusculary, transdermally, pulmonary, subcutaneously, intradermally, intratechally, through the buccal-, anal-, vaginal-, conjunctival-, or intranasal tissue, by inoculation into tissue, by an implant, or orally.

40. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 50 mg per week.

41. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 100 mg per week.

42. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 250 mg per week.

43. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 500 mg per week.

44. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 750 mg per week.

45. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 1000 mg per week.

46. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 1250 mg per week.

47. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 1500 mg per week.

48. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 2000 mg per week.

49. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 2500 mg per week.

50. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 5000 mg per week.

51. A method according to claim 1, comprising administration during at least 1 day.

52. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 10 mg/kg body weight.

53. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 50 mg/kg body weight.

54. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 100 mg/kg body weight.

55. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 200 mg/kg body weight.

56. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 500 mg/kg body weight.

57. A method according to claim 7, wherein the trimeric complex is administered in an amount of at least 1000 mg/kg body weight.

58. A method according to claim 1, comprising administering a dose of said pharmaceutical composition once a week.

59. A method according to claim 1, comprising administering a dose of said pharmaceutical composition once every second week, once every third week, or once every fourth week.

60. A method for the prevention and/or treatment of a disease or condition, said method comprising transfecting a cell population with a nucleic acid sequence encoding an apolipoprotein protein construct as defined in claim 1, and subsequently transferring said cell population to an individual in need thereof.

61. A method according to claim 60, wherein the cell population comprises macrophages.

62. A method according to claim 60; wherein the cell population comprises liver cells.

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