RECOMBINANT FACTOR H AND VARIANTS AND CONJUGATES THEREOF

Abstract

The present invention relates to recombinant factor H and variants and conjugates thereof and methods of their production, as well as uses and methods of treatment involving the materials.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to recombinant factor H and variants and conjugates thereof and methods of their production, as well as uses and methods of treatment involving said materials.

BACKGROUND OF THE INVENTION

[0002] An increasing body of evidence suggests that the complement system regulatory glycoprotein, factor H (FH), if produced in sufficient quantities and endowed with appropriate pharmacokinetic and pharmacodynamic properties, would serve as a new biotherapeutic agent. This agent could prevent development of age-related macular degeneration (AMD) in genetically susceptible individuals and facilitate treatment in those with AMD and two life-threatening kidney conditions known as atypical haemolytic uraemic syndrome (aHUS) and dense deposit disease (DDD). More speculatively, this agent could have beneficial effects in the treatment or prevention of numerous other diseases in which inadequate complement regulation contributes to aetiology or symptoms.

[0003] However current attempts to produce FH through over-expression of a gene in recombinant cells have failed to yield the quantities that would be required for therapy, while purification from human plasma of sufficient quantities of the appropriate variants of FH has logistical and technical difficulties and carries health risks. There are urgent unmet clinical and commercial needs for multiple-gram quantities of biotherapeutic-grade recombinant versions of FH with minimal immunogenicity, an extended half-life and maximal efficacy.

[0004] Links between polymorphisms in FH and susceptibility to disease have been well documented and are reviewed, for example in: Opportunities for new therapies based on the natural regulators of complement activation. Brook E, Herbert A P, Jenkins H T, Soares D C, Barlow P N. Ann N Y Aced Sci 2005 1056:176-88; Complement factor H: using atomic resolution structure to illuminate disease mechanisms. Barlow P N, Hageman G S, Lea S M. Adv Exp Med. Biol. 2008 632:117-42; Translational mini-review series on complement factor H: renal diseases associated with complement factor H: novel insights from humans and animals. Pickering M C, Cook H T. Clin Exp Immunol 2008 151:210-30; Translational mini-review series on complement factor H: genetics and disease associations of human complement factor H. de Cordoba S R, de Jorge E G. Clin Exp Immunol. 2008 151:1-13.

[0005] Since these reviews were published numerous further published findings have broadened the scope of potential targets for FH-based therapies. Two recent examples establish an association between the FH gene (CFH) polymorphism (Y402H) and susceptibility to cardiovascular disease (CVD). Koeijvoets et al. (Complement factor H Y402H decreases cardiovascular disease risk in patients with familial hypercholesterolaemia. Koeijvoets K C, Mooijaart S P, Dallinga-Thie G M, Defesche J C, Steyerberg E W, Westendorp R G, Kastelein J J, van Hagen P M, Sijbrands E J. Eur Heart J. 2009 30:618-23. showed that amongst patients with severely increased risk of early-onset CVD due to hypercholestrolaemia, the Y402 CFH variant was inversely associated with susceptibility to CVD suggesting that CFH modifies the risk of CVD. In a study by Buraczynska et al. (Complement factor H gene polymorphism and risk of cardiovascular disease in end-stage renal disease patients.Buraczynska M, Ksiazek P, Zukowski P, Benedyk-Lorens E, Orlowska-Kowalik G. Clin Immunol. 2009; 132:285-90) of end-stage renal failure in patients on dialysis, multivariate logistic regression analysis showed that the Y402H genotype is independently associated with cardiovascular co-morbidity; with homozygosity for the H402 allelebeing associated with an odds ratio of 7.28 (95% Cl 5.32-9.95). In another recent development, Moreno-Navarrete et al. (Complement Factor H is expressed in adipose tissue in association with insulin resistance. Moreno-Navarrete J M, Martinez-Barricarte R, Catalan V, Sabater M, Gomez-Ambrosi J, Ortega F J, Ricart W, Bluher M, Fruhbeck G, de Cordoba S R, Fernandez-Real M J. Diabetes 2009: Epub October 15) showed that FH is expressed in adipose tissue in association with insulin resistance, suggesting a link between the alternative pathway of the complement system, obesity and metabolic disorders.

[0006] Data for the likely efficacy of FH in treatment is already very strong, and has precipitated numerous disclosures, patent applications and company start-ups. US2007/0020647 discusses the expression of human CFH in a variety of eukaryotic and prokaryotic protein-overproduction vectors and in mammalian cell lines, but only explicitly exemplifies expression in the human lung carcinoma cell line A549. The quantities of recombinant protein obtained from this cell line are not disclosed, but based on precedent and in the absence of any evidence to the contrary the amounts are expected to be inadequate for therapeutic purposes. WO2007/038995 describes the use of human factor H to treat aHUS. The patent application mentions the use of recombinant FH without providing significant details about the methods of production of recombinant FH, but is focused on purification of FH from human plasma.

[0007] Thus although the above two documents disclose the idea of using recombinant FH therapeutically, neither document actually teaches the large-scale production of recombinant FH that is absolutely essential for its therapeutic application; as shown herein, this is not a straightforward task.

[0008] Successful manufacture of larger amounts (greater than 10 mg) of pure recombinant full-length FH with preserved functional activities has not previously been reported in the scientific or patent literature. Indeed, in the limited data supporting the patents discussed above, the authors demonstrated capability of producing only minute quantities (less than about 1 mg) of recombinant FH and did not provide evidence that they had purified or characterised this material. Furthermore, literature reports likewise allude to sub-milligram quantities of recombinant FH from insect and mammalian cells (e.g. Biologically active recombinant human complement factor H: synthesis and secretion by the baculovirus system. Sharma A K, Pangburn M K. Gene 1994 143:301-2; Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome. Sanchez-Corral P, Perez-Caballero D, Huarte O, Simckes A M, Goicoechea E, Lopez-Trascasa M, de Cordoba S R. Am J Hum Genet. 2002 71:1285-95.) or to expression of fragments, only, of the FH molecule (e.g. Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations. Hocking H G, Herbert A P, Kavanagh D, Soares D C, Ferreira V P, Pangburn M K, Uhrin D, Barlow P N. J Biol Chem 2008 283:9475-87).

[0009] Ormsby, R. J. et al., Expression of human factor H in the methylotrophic yeast Pichia Pastoris. Molecular Immunology Vol 35, p. 353, 1998 Abstract 92. This paper uses a Pichia pastoris production system to express a FIVE (5) complement control protein (CCP) fragment of Factor H, not the full length TWENTY (20) CCP Factor H protein, which is the subject of present patent application.

[0010] Ripoche, J. et al., The complete amino acid sequence of human complement Factor H. Biochemical Journal, Vol 249: 593-602, 1988. This paper describes the full length human factor H nucleotide sequence (and hence the amino acid sequence) and was obtained by sequencing three overlapping cDNA clones spanning the Factor H gene. However, it does not describe how to clone the gene such that it is possible to express functional human Factor H protein.

[0011] EP1336618 describes using full length or fragments of porcine Factor H as a soluble complement regulator, for use as a therapeutic. It is suggested that porcine factor H could be purified from pig plasma or as exemplified in this patent, made recombinantly using Baculovirus. However, no quantification of the amount of full length porcine factor H from a standard fermentation nor any functional data for the full length protein (rather than only fragments) is shown. However, there is no disclosure or teaching of how to express functional human Factor H.

[0012] The use of porcine Factor H naturally carries the risk of infection with cross-species zoonotic infections. Moreover, there is not complete DNA sequence or amino acid homology between human factor H and porcine factor H (62% homology Hegasy G. A. et al., Pig complement regulator factor H: molecular cloning and functional characterization. Immunogenetics. 2003 October; 55(7):462-71). It is therefore very likely autoantibodies to porcine Factor H would be made, which would again limit therapeutic usage.

[0013] WO 2008/135237 describes use of a therapeutic which combines a short consensus repeat (SCR) of Factor H with a pathogen recognition binding molecule e.g. an antibody. It specifically mentions use of fragments/peptide chains of less than 100 amino acids (<2 SCRs). It does not suggest use of a full length Factor H molecule with a pathogen recognition binding molecule. Also, its focus is for the use of treating infections or for cancer, not renal or opthalmological diseases.

[0014] Currently, FH-replacement clinical therapy is achieved by means of infusing donated pooled plasma, of which FH is only one of many protein components. It is not possible clinically to routinely obtain plasma containing only the FH Y402 allotype (which is protective against AMD); when purified in bulk from pooled plasma, FH is heterogeneous in terms of both its heterotypic and glycoform variations and hence this material is ill-suited for therapy; antibody-affinity based purification methods generally yield only small amounts (a few mg at most) of material that can be enriched only for a single variant at a specific site of variation (e.g. for Y402) but will be heterogeneous with respect to other polymorphic sites (e.g. V62I). Any use of plasma-purified human proteins would in any case may carry unacceptable risks, of infection with both unknown viral and prion proteins, and of sensitisation to contaminating plasma components, when used on the repetitive basis proposed for AMD, aHUS and DDD therapies.

[0015] It is therefore amongst the objectives of the present invention to obviate and/or mitigate at least one of the aforementioned obstacles to therapeutic use of FH.

SUMMARY OF THE INVENTION

[0016] The invention is based on work carried out by the present inventors towards providing high-yield production of versions of FH tailored for animal and human trials and therapeutic applications, which is based on the use of codon-optimised chemically synthesised genes that are transfected into, for example and preferably, Pichia pastoris followed by expression in a fermentor and purification using a sequence of chromatographic procedures.

[0017] In a first aspect there is provided a process for making recombinant mammalian FH, said process comprising the steps of:

[0018] expressing in a chosen host organism a codon-optimised nucleic acid sequence which encodes said mammalian FH or variants thereof and which nucleic acid sequence has been codon optimised for expression in a chosen host organism and inserted into an appropriately designed vector; in order to obtain said mammalian FH or variants thereof.

[0019] Conveniently, the codon-optimised nucleic acid sequence can initially be chemically synthesised rather than cloned and mutagenised in order to generate the necessary codon optimisation. In accordance with the present invention it is possible to produce large quantities of recombinant mammalian FH and its variants hitherto not possible using the previously described techniques. Typically the methods of the present invention may produce protein yields of at least 0.5 mg of recombinant FH (or its variants) per liter of culture medium, such as at least 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg or 500 mg per litre of culture medium. It will therefore be appreciated that it is possible following the methods of the present invention, when using industrial-scale fermentors, to produce hundreds of milligrams or gramsoreven kilogram-quantities of recombinant FH and variants thereof, which was simply not possible using conventionally cloned recombinantly expressed FH.

[0020] The above process may further comprise purifying said proteins from the cell and/or culture medium in which the cell is grown. Purification may typically involve the use of chromatographic methodologies, such as fast-protein liquid chromatographic or high-performance (pressure) liquid chromatographic techniques known in the art. For example, the nucleic-acid sequence may be designed to encode a secretion-signal sequence of amino-acid residues fused to the N-terminus of FH so that FH is secreted into the media (whereupon said signal-sequence peptide is cleaved off) and thereby it is separated from intracellular P. pastoris proteins at the outset. In a subsequent purification step, crude material may, for example, be loaded onto an affinity chromatography column, such as a heparin-sepharose column equilibrated in phosphate-buffered saline (PBS), and eluted by application of a gradient, over multiple column volumes, to PBS substituted with high salt (e.g.1 M NaCl); in a further step, FH-containing fractions from the previous step may be loaded onto, for example, an ion-exchange resin-containing column, such as a GEHealthcare-supplied MonoQ column that has been equilibrated in 20 mM glycine buffer (typically pH 9.5, 150 mM NaCl), and then eluted with a gradient, over many column volumes, with the equilibration buffer at the same pH but substituted with high salt (e.g. 1 M NaCl).

[0021] The preferred choice of host organism is Pichia pastoris on the grounds that no re-folding of the expressed protein is required, the protein may be secreted into the media and therefore easily accessible, and specific glycoconjugates or non-natural amino acid residues may be incorporated into the recombinant product; but other prokaryotic (e.g. Escherichia coli) and eukaryotic (e.g. Sacchyromyces cerevisiae) host organisms may also be envisaged.

[0022] The mammalian FH referred to may be human FH or FH from another primate or other mammalian FH, such as that from mouse, rat, hamster, rabbit, dog, horse, cow, pig, sheep, camel, cat, guinea pig, or the like.

[0023] The deoxyribonucleic nucleic acid (DNA) sequence may comprise unique restriction endonuclease sites at the 5' and 3' ends of the nucleic acid, to facilitate cloning into an appropriately restricted expression vector. Preferred restriction sites are PstI, BamHI, NotI and XbaI, although others may easily be envisaged by the skilled addressee.

[0024] The nucleic acid sequence encoding FH may relate to one of a number of wild-type sequences (known in the art as polymorphic variants) or may be a mutant sequence. The sequence may comprise one or more single-nucleotide polymorphisms known in the art. US 2007/0020647, for example, describes many polymorphisms that have hitherto been identified in the human CFH (the contents of which are hereby incorporated by way of reference) and more such polymorphic variants may be discovered in the future; one or more of these may readily be incorporated into the codon-optimised nucleic acid sequence. Preferred single-nucleotide polymorphisms that may be incorporated, individually or in combination, into the codon-optimised nucleic acid sequence could code for the following variations in the protein sequence: Ile62 (rather than Val), Tyr402 (rather than H is), Glu936 (rather than Asp) and/or Arg1210 (rather than Cys) (all numbers refer to the sequence of the encoded protein prior to cleavage of the signal sequence (Swiss-Prot: P08603.4)). Such single-nucleotide polymorphisms and haplotypes have been reported to be associated with a lower-than-average risk of developing AMD (Hageman G S et al. A common haplotype in the complement regulatory gene factor H(HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci USA 2005 102:7227-32; Klein R J et al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005 308:385-9; Edwards A O et al. Complement factor H polymorphism and age-related macular degeneration. Science 2005 308:421-4; Haines J L et al. Complement factor H variant increases the risk of age-related macular degeneration. Science 2005 308:419-21; Hageman G S et al. Extended haplotypes in the complement factor H(CFH) and CFH-related (CFHR) family of genes protect against age-related macular degeneration: characterization, ethnic distribution and evolutionary implications. Ann Med 2006 38:592-604). Alternatively or additionally, mutant sequences may be designed to specifically alter the FH polypeptide sequence, for example to include one or more natural (encoded) or non-naturally encoded variant amino acids as described in more detail herein below.

[0025] The conjugate refers to a molecule that consists of a polypeptide corresponding to FH or a variant of FH to which is covalently attached, normally via one or more amino-acid residue side-chains, to a chemical moiety or moieties intended to improve the biotherapeutic properties of said molecule.

[0026] The attached moieties could include: natural polymers such as glycosaminoglycans and their derivatives or polysialic acids, dextran (-1,6 polyglucose), dextran (-1,4 polyglucose), hyaluronic acid, and chitosans; unnatural polymers such as any of a large family of linear or branched polyethylene glycols, polyether polyols, N-(2-hydroxypropyl) methacrylamide copolymers, poly(vinylpyrrolidone), poly(ethyleneimine), or linear polyamidoamines; or pseudosynthetic polymers, such as poly(L-lysine), poly(glutamic acid), poly(malic acid) and poly(aspartamides) (see for example The dawning era of polymer therapeutics. Duncan R. Nature Reviews Drug Discovery 2003 2:347-360).

[0027] Rather than conventional gene cloning and expression, the present invention is based on an initial chemical synthesis of the codon-optimised DNA molecules encoding said FH (and variants thereof), using gene design and synthesis techniques in the art (e.g.Gene composer: database software for protein construct design, codon engineering, and gene synthesis. Lorimer D, Raymond A, Walchli J, Mixon M, Barrow A, Wallace E, Grice R, Burgin A, Stewart L. BMC Biotechnol. 2009 9:36). In this manner, the codon-optimised nucleic acid is synthesised de novo prior to cloning into a suitable expression vector. Conventional site-directed mutagenesis techniques known in the art to carry out codon optimisation of the FH gene would be unfeasibly time-consuming, if not impossible due to the high risk of introducing additional mutational variations during the requisite repeated rounds of site-directed mutagenesis. However, site-directed mutagenesis may be used following cloning of the synthetic codon-optimised CFH, in order to accomplish one or a combination of site-specific mutations in the product

[0028] Codon optimisation is carried out in order to enhance the expression levels of the mammalian FH and its variants in the desired host organism, such as P. pastoris. Said optimisation involves one or more of the following: adapting codon bias to match that of the chosen host organism; avoiding regions of high (>80%) or low (<30%) GC content; minimising any potential internal TATA boxes, chi-sites and ribosome-entry sites; minimising AT-rich or GC-rich stretches of sequence, avoiding repeat sequence and RNA secondary structures, minimising any (cryptic) splice-donor and/or splice-acceptor sites; and ensuring any desired restriction endonuclease sites are only found at the extreme 5' and 3' ends of the nucleic acid to facilitate cloning. Preferably all of the above considerations are taken into account when optimising the nucleic acid sequence. The skilled addressee is able to make such modifications to the original FH sequence based on prior knowledge in the art in relation to the codon bias of the chosen host and other teachings (e.g.Codon bias and heterologous protein expression. Gustafsson C, Govindarajan S, Minshull J. Trends Biotechnol 2004 22:346-53). Certain companies such as Geneart (Regensburg, Germany), GeneScript (Piscataway, N.J., USA) and DNA2.0 (Menlo Park, Calif., USA) provide a service for optimising and synthesising nucleic acid sequences that are tailored for expression in a specified host organism.

[0029] In a preferred embodiment, the DNA sequence encoding mammalian FH is a CFH sequence which has been optimised for expression in the host, P. pastoris. A P. pastoris codon-optimised human CFH sequence (encoding for Y at position 402, I at position 62 and E at position 936) is compared to the wild-type cDNA sequence in FIG. 1. It will be appreciated that this codon-optimised sequence may be varied in order to still further optimise the sequence for overproduction in P. pastoris. Moreover, the sequence may be easily varied in order to allow for expression of various allotypes. Moreover, certain nucleotide bases may be changed in order to specifically alter the amino-acid residue sequence of the FH protein. For instance, certain amino-acid residues may be replaced with, for example, alternative amino-acid residues that may be rare or non-naturally occurring amino-acid residues, so as to allow for the generation of recombinant FH proteins with one or even a combination of modifications leading to: altered glycosylation patterns; reduced immunogenicity; enhanced plasma half-life; and/or site-specific conjugation with moieties designed to improve pharmacokinetic and/or pharmacodynamic properties. It will be appreciated that all such modifications can be carried out whilst taking account of any codon optimisation considerations.

[0030] Thus, in a further aspect, the present invention provides a nucleic acid sequence capable of expressing a FH polypeptide or variant thereof, the nucleic acid sequence being codon optimised for expression in a host organism, such as P. pastoris. There is also provided a mammalian FH polypeptide or variant thereof, obtained from a nucleic acid sequence according to the present invention.

[0031] Preferably the sequence is codon optimised for expression by P. pastoris, in which case the nucleic acid sequence may be the codon-optimised human sequence shown in FIG. 1 or any of the sequences represented in FIGS. 5A and 5B, or be substantially similar to them. By substantially similar is understood that the sequence is greater than 70%, 75%, 80%, 85%, 90%, 95% or even 99% identical to the sequence shown in FIG. 1 or 5A or 5B.

[0032] The present invention also relates to vectors which include a codon-optimised FH-encoding DNA sequence of the present invention, host cells which are genetically engineered with said recombinant vectors, and the production and purification of the encoded FH and FH-like polypeptides by recombinant techniques, and the conjugated products of said polypeptides.

[0033] Recombinant constructs may be introduced into host cells using well-known techniques such as infection, transduction, transfection, transvection, electroporation and transformation. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

[0034] The polynucleotides of interest may be contained within a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in the form of a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[0035] Preferred, are vectors comprising cis-acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

[0036] In certain preferred embodiments in this regard, the vectors provide for specific expression and may be inducible and/or cell type-specific. Suitable vectors include those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.

[0037] Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, for example vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.

[0038] The DNA insert should be operatively linked to an appropriate promoter. Known bacterial promoters suitable for use in the present invention include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the phage lambda P_R and P_L promoters and the tac and trp promoter. Suitable eukaryotic promoters include the cytomegalovirus immediate early promoter, the herpes simplex virus thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral long terminal repeats (LTRs), such as those of the Rous sarcoma virus and metallothionein promoters, such as the mouse metallothionein-I promoter. Promoters specific to P. pastoris include alcohol oxidase 1(AOX1), AOX2 (both methanol inducible), CUP1 (copper inducible), GAP (glycerol inducuble, constitutively active on various carbon sources), FLD1 (formaldehyde dehydrogenase gene)http://faculty.kgi.edu/cregg/PP strains.htm-pblhisix, PEX8 (moderate promoter)http://faculty.kgi.edu/cregg/PP strains.htm-pblhisix, YPT1 (moderate promoter, constitutively active on various carbon sources)http://faculty.kgi.edu/cregg/PP strains.htm-pblhisix, DAS1 (dihydroxyacetone synthase)http://faculty.kgi.edu/cregg/PP strains.htm-pblhisix, ADH1 (alcohol dehydrogenase)http://faculty.kgi.edu/cregg/PP strains.htm-pblhisix and PGK1 (3-phosphoglycerate kinase). Other suitable promoters will be known to the skilled artisan, see for example Cereghino and Cregg, 1999, Current Opinion in Biotechnology, 10, p422-427.

[0039] The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome-binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation-initiating AUG at the beginning and a termination codon appropriately positioned at the end of the nucleic acid sequence to be translated. It is facile, using synthetic genes, to optimise all of these features of the insert to maximise gene-expression levels and recombinant-protein yields.

[0040] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include e.g. dihydrofolate reductase or neomycin or zeocin resistance for eukaryotic cell culture and e.g. tetracycline or ampicillin-resistance genes for culturing in E. coli and other bacteria.

[0041] Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells like P. pastoris, Kluyveromyceslactis and Sacchyromyces cerevisiae; insect cells such as Drosophila melanogastor S2 and Spodoptera frugiperda 9 cells; animal cells such as Chinese hamster ovary, COS and Bowes melanoma cells; and plant cells. Appropriate culture media and conditions for the above-described host cells are known in the art. Most preferably the host organism is the methylotropic yeast P. pastoris. Strains of P. pastoris that have been metabolically engineered so that they attach mammalian or human-like N-glycans may be preferred, see Wildt and Gerngross, 2005, Nature Reviews, 3, p119-128, Li et al, 2006, Nature Biotechnology, 24, p210-215, Cereghino, et al, 2002, Current Oinion in Biotechnology, 13, p329-332.

[0042] Vectors preferred for use in bacteria include pA2, pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNHb8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Vectors preferred for use in P. pastoris include pPIC9K, pHIL-D2, pHIL-S1, pPIC3.5K, pGAPZ, pGAPZalpha, pPICZalpha-A, pPICZalpha-B, pPICZalpha-C, pPICZalpha-E, pPICZalpha-E/Uni, pPIC3.5, pPIC9, pPICZ-A, pPICZ-B, pPICZ-C, pPICZ-E from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

[0043] As indicated, introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis L G G et al., Basic Methods in Molecular Biology, (2n^d Ed., McGraw-Hill, 1995).

[0044] As indicated, transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at by 100 to 270, the cytomegalovirus early-promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0045] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous. Examples of such sequences that may be used in P. pastoris include the native human or mouse (or other mammalian) FH-secretion signals and the yeast alpha-mating factor.

[0046] The polypeptide of interest may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. Thus, for instance, a region of additional amino-acid residues, particularly charged amino-acid residues, may be added to the N terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be fused to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Additions of peptide moieties to polypeptides in order to engender secretion or excretion, to improve stability and to facilitate purification, amongst others, are familiar and routine techniques in the art.

[0047] The FH protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion-exchange or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, reverse-phase chromatography, size-exclusion chromatography and lectin chromatography. Most preferably, heparin-affinity is followed by ion-exchange chromatography.

[0048] It will be recognised in the art that the amino-acid residue sequence of aFH polypeptide may be selectively varied without having a significantly detrimental effect on the structural integrity or functional properties of the protein. If such differences in sequence are contemplated, it should be remembered that there are regions of the protein that are critical to its biological activity. There will also be residues that are critical to the folding of the protein or for stabilisation of its folded structure. Some residues serve as glycosylation sites, recognised by enzymes that covalently attach glycans to, for example, Asn side-chains. In general, it may be possible to safely replace residues that contribute directly or indirectly to structure or function by other residues that are chemically similar (this is known as a conservative substitution). In the cases of amino-acid residues that contribute neither to structural integrity nor to functional sites, it may be possible to safely replace such a residue with an amino-acid residue of a different chemical nature (a non-conservative replacement).

[0049] Thus, the invention further includes variations of the FH polypeptide which variants show substantially FH-like biological activity. Variants might include conservative substitutions (for example, substituting one hydrophilic residue for another, or one hydrophobic residue for another), but would be unlikely to include replacements of strongly hydrophilic residues for strongly hydrophobic ones (or vice versa). Variants might include conservative substitutions within N-glycosylation sites that result in loss of such sites. Variants may also include deletions of one or more of the 20 protein domains within the FH molecule. For example, deletion of one or a combination of domains [such as 2, 3, 4, 5, 6, 7, 8, 9, or 11 domains] between and including domains 8 and 18 would be unlikely to have a detrimental effect on the functionally critical individual binding sites located in domains 1-4, 6-7 (or 6-8) and 19-20. Variants could also include deletions of one or a combination of domains from the region of FH between and including domains 5-18 since this would preserve C3b-binding sites (1-4, and 19-20) and one (in 19-20) of two cell surface-recognition sites within FH (see e.g. A new map of glycosaminoglycan and C3b-binding sites on factor H. Schmidt C Q, Herbert A P, Kavanagh D, Gandy C, Fenton C J, Blaum B S, Lyon M, Uhrin D, Barlow P N. J Immunol, 2008, 181:2610-9) and might enhance functional activity by optimising the spatial positioning, or flexibility of the connection, between these binding sites. Variants might also include hybrids, in which, for example one or more deleted domains from the domains 8-18, or 5-18, regions of FH are replaced with one or more similar domains derived from other proteins, for example from complement receptor type I or type II; alternatively they might be replaced by one or more dissimilar domains derived from a wide range of other proteins such as proteins of the extracellular matrix or the clotting or complement cascades.

[0050] Typically seen as conservative substitutions are the replacements, one for another, amongst the aliphatic amino-acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements amongst the aromatic residues Phe and Tyr. Non-conservative substitutions could include substitutions with both naturally encoded amino-acid residues and a non-naturally encoded (unnatural) amino-acid residue. The unnatural amino-acid residue could be one that serves as a site-specific attachment sites for conjugation with chemical moieties (such as polyethylene glycols (PEGs) and other polymers), or with biochemical groups (such as glycans) that enhance the therapeutic efficacy of FH.

[0051] As indicated in detail above, further guidance concerning which aminoacid changes are likely to be phenotypically silent (i.e. are not likely to have a significant deleterious effect on a function) can be found in Bowie, et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).

[0052] Also of interest are substitutions that prevent aggregation or minimise proteolysis. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because aggregates can be immunogenic (see, e.g. Pinckard et al., Clin Exp. Immunol, 1967, 2:331-340; Robbins et al., Diabetes, 1987, 36:838-845; Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems, 1993, 10:307-377). Aggregation may be minimised by changing surface residues, for example removing hydrophobic patches (by substituting hydrophobic residues with polar ones) or by changing the electrostatics at the surface by charge-reversal (e.g. by substituting Asp for Arg or Glu for Lys) or deletion (e.g. substituting Ser for Asp). Proteolysis results in a loss of the target protein thus lowering yield and also makes purification more difficult. Proteolysis may be reduced by recognition of proteolytic sites via computational prediction or empirical means and conservative substitutions therein.

[0053] Possible modifications of particular relevance to mammalian FH include mutating one or more Asn residues to Gln residues in order to minimise glycosylation of the FH protein. Alternatively one or even two Asn residues of the FH protein may be replaced by any of a large number of unnatural amino-acid residues, such as p-(propargoxy)-phenylalamine (pPpa) residues (Expanding the genetic repertoire of the methylotrophic yeast Pichia pastoris. Young T S, Ahmad I, Brock A, Schultz P G. Biochemistry 2009 48:2643-53). Such an unnatural residue could be further modified by PEGylation or sialylation techniques known in the art.

[0054] Thus, in a further aspect, the present invention provides a recombinantly expressed variant of mammalian, especially human, FH obtained from a codon-optimised nucleic acid wherein the variation comprises one or more amino-acid residue substitutions designed to modulate one or more biological properties of said FH variant as compared to a native FH.

[0055] It will be understood that said amino acid substitution(s) do not relate to polymorphic changes to the FH protein, as known in the art. Said substitution(s) may result in modulation of, for example, immunogenicity and/or a physiological property of said FH variant as compared to a native FH. Exemplar modifications include substituting one or more Asn residues for another amino acid residue, such as Gln, or a non-naturally occurring amino acid residue, such as pPpa, in order to vary the glycosylation state of said FH variant and/or allow further modification of said FH variant using chemistry known in the art in order to allow the variant to be specifically modified at said substituted sites by a molecule such as PEG or polysialyl chains.

[0056] Thus, the target FH-like polypeptide may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues is conjugated with another molecule or includes a substituent group; or (iii) one in which the mature polypeptide may be covalently linked to another compound or compounds, such as a compound to increase the half-life of the polypeptide (for example, PEG or polysialic acid); or (iv) one in which additional amino acid residues are fused to the mature polypeptide, such as an IgG Fc fusion-region peptide or leader or secretion-signal sequence or a sequence which is employed for purification of the mature polypeptide or a pro-protein sequence; or (v) one with an altered (compared to the native glycoforms of FH) pattern of attached glycans due to substitutions within glycosylation sites or introduction of new glycosylation sites (or unnatural amino acids suitable for chemical conjugation with glycans) or employment of strains of P. pastoris with engineered glycosylation pathways; or (vi) domain-deletion or hybrid variants in which domains have been removed from the central portion of FH and may or may not have been substituted with homologous or other domains from other proteins. Such fragments, derivatives, conjugates and analogues are deemed to be within the scope of those skilled in the art from the teachings herein.

[0057] The recombinantly expressed mammalian FH polypeptides and variants of the present invention may find a variety of applications. For example the polypeptides/variants may be used therapeutically to treat or prevent age-related macular degeneration (AMD) or to prevent or slow the progression of this disease, in genetically susceptible individuals and facilitate treatment in those with AMD, as well as in the treatment/prevention of two life-threatening kidney conditions known as atypical haemolytic uraemic syndrome (aHUS) and dense deposit disease (DDD). The recombinantly expressed mammalian FH of the present invention could have beneficial effects in the treatment or prevention of numerous other diseases or pathologies in which inadequate complement regulation contributes to aetiology or symptoms, for exampleAlzheimer's disease, ischemia, pre-eclampsia, early pregnancy loss, sepsis, multiple sclerosis, system lupus erythematosus and transplant rejection. See for example, Ischaemia-Reperfusion Injury: (Shah K G et al, J Surg Res. 2010 163 1:110-117; Yang J et al, Ann Surg. 2009 249 2:310-317; Zhang F et al, Regul Pept. 2009 8 52(1-3):82-87, Organ Transplantation: Atkinson C, et al, J Immunol. 2010 185 11:7007-7013., Early Pregnancy Loss: Lynch A M, et al, Obstet Gynecol. 2011 117 1:75-83 and Pre-eclampsia Qing X et al, Kidney Int 2010 October 13. [Epub ahead of print].

[0058] The recombinant FH polypeptides and variants in accordance with the present invention may also find application in research and also in kits and the like.

[0059] Particularly preferred FH molecules and variants according to the present invention are described in detail below.

DETAILED DESCRIPTION

[0060] The present invention will now be further described by way of example and with reference to the figures which show:

[0061] FIG. 1 shows DNA sequence (Swiss-Prot: P08603,4) of native human FH; Sequence of P. pastoris codon-optimised human FH of the present invention; and an alignment of the wild-type (cDNA-derived) and codon-optimised FH gene sequences.

[0062] FIG. 2 shows western-dot-blot results from non-codon optimised FH gene expression.

[0063] FIGS. 3A-3J show_production and characterisation of recombinant complement factor H:

[0064] FIG. 3A--Elution from an anion-exchange column (MonoQ) (A²⁸⁰ in milli-absorbance units on left-hand y-axis) with a salt gradient (20 mM glycine buffer, pH 9.5, 0.12-1 M NaCl; conductivity on right-hand y-axis).

[0065] FIG. 3B--Fractions eluted from the MonoQ column (see FIG. 3A) were subjected to SDS-PAGE and protein bands were visualised with Coomassie blue. Lanes 1-8 (reducing conditions--i.e. no disulfides present) correspond to elution volumes 23-30. No significant "clipping" of the polypeptide chain is evident. Lane 9 contains molecular weight markers (MW) as indicated on the right-hand side. Lanes 3', 4' and 5' correspond to lanes 3, 4 and 5 but were run under non-reducing conditions; the faster migration of bands in lanes 3', 4', and 5' (compared to lanes 3, 4 and 5) is typical for proteins that contain disulfide bonds.

[0066] FIG. 3C--Two antibodies that recognise epitopes within the C-terminal CCP modules (domains) of FH, were used in western blots. Plasma FH (left lane) andrecombinant rFH (middle lane) were detected with (i) MAb-SC47686_L20/3 or (ii) Mab-Abnova-0167. MW=molecular weight markers--see right-hand side of gel (ii).

[0067] FIG. 3D--The abilities of FH (lanes 1-5) and rFH (lanes 7-11) to act as cofactors for factor I-catalysed cleavage of C3b to iC3b were assessed by visualising the 43-kDa and 68-kDa proteolytic fragments of the a'-chain using SDS-PAGE followed by Coomassie blue staining. Incubation times were 0 to 30 minutes, as indicated. Both versions of FH have similar activities in this semi-quantitative assay such that the a'-chain of C3b is completely processed within five minutes. MW=molecular weight markers of (from top) 250, 150, 100, 75, 50, 37, 25 and 20 kDa.

[0068] FIG. 3E--For comparison with FIG. 3D, the cofactor activity of soluble complement receptor type 1 (sCR1), at the same concentration was followed over the same time intervals. Note that (in agreement with literature) SCR1, but (from FIG. 3D) neither rFH nor plasma-purified FH, promoted the further degradation of the α'-chain to C3dg and a 30-kDa fragment. MW, as in FIG. 3D.

[0069] FIG. 3F--Surface plasmon resonance was used to monitor formation of the C3bBb (convertase) complex as factor D and factor B were flowed together over C3b that was amine-coupled to a CM5 (Biacore) sensor chip. The subsequent decline in response reflects decay of the complex as Bb is released from the chip surface. The rate of decay is accelerated by initiating (in this case 210 s into the natural decay process) a flow of reference FH or rFH. At similar concentrations (0.5 μM), rFH is a more effective decay accelerator in this assay than plasma-purified FH. The control proteins, BSA and FH modules 19-20, have no effect on decay.

[0070] FIG. 3G--(i) and (ii)--Use of SPR to measure affinity of (i)rFH and (ii) plasma-purified FH for C3b coupled to aCM5 sensor chip (Biacore). Duplicate sensorgrams are shown for a concentration series (5.4 μM, 1.0 μM, 0.5 μM, 0.1 μM) flowed over 1540 response units of immobilised C3b. (iii) and (iv)--Plots of response units versus (iii)rFH or (iv) plasma-purified FH concentrations for two different flow cells with either 1540 RUs (lower curve in each plot) or 3030 RUs (upper curve in each plot) of C3b. The dashed vertical line indicates the K_D fitted in each case to both plots simultaneously, and yielding 1.4 μM for rFH and 2.9 μM for plasma-purified FH.

[0071] FIG. 3H--The candidate recombinant FH (peaks a and c correspond to double-charged and single-charged species, respectively) and an internal standard (IgG₁; peaks b and d correspond to double-charged and single-charged species, respectively) were analysed on a MALDI-ToF mass spectrometer.

[0072] FIG. 3I--Dynamic light scattering was performed on rFH in PBS at a concentration of 1 mg/ml.

[0073] FIG. 3J--Sheep erythrocytes were incubated in physiological buffer, with 1.5 μM F H modules 6-8 (negative control), 0.4 μM plasma-purified FH or 0.4 mM rFH prior to exposure (for 20 minutes at 37° C.) to human serum that had been depleted of FH. TIe reaction was quenched and A₄₁₂ was measured. The results shown were the average (plus or minus standard deviation) of four experiments.

[0074] FIG. 4A shows a schematic representation of human factor H (FH) showing certain SNP's and the eight N-linked glycans. FIG. 4B shows schematic representations of vector (plasmid) maps designed such that various FH molecules and variants can be prepared in accordance with the present invention. All except vector 4 (based on pPICZα-B) are based on pPIC3.5K. Vector numbers 1-3 and 11 incorporate DNA for the human secretion signal peptide (hum. signal pept.) while vector numbers 7, 9 and 10 incorporate the mouse equivalent. The other four vectors incorporate DNA for the yeast alpha-factor peptide with (vector number 4) or without (vectors 5, 6 and 8) EA dipeptides. The encoded variants of FH (sequences in FIGS. 5A and 5B) are indicated--the protective (prot.) and at-risk haplotypes are detailed in the text; "all-Q" and "one amber Q" or "two amber 0" refer to substitutions of Asn residues for Gln and one or two pPa residues (for example), respectively, as described in the text; "delta 10-15" indicates removal of FH domains 10-15 as described in the text; K/R indicates substitution of lysines and arginines with glutamines as described in the text.

[0075] FIGS. 5A and 5B are a summary of DNA sequences encoding (a) human and (b) mouse FH variants that have been inserted into vector numbers 1-11.

[0076] FIG. 6 illustrates the expression of two recombinant variants of FH. The sample of "all-Q" mutant of rhFH (left-hand gel) migrates as a single band during SDS-PAGE under reducing (R) and non-reducing (NR) conditions (stained by Coomassie blue). Endo H_f (77 kDa) treatment causes no change in migration rate. This is consistent with the "all-Q" mutant having no N-glycosylation sites and being glycan-free. For comparison (middle gel), rhFH (prior to purification) migrates as a fuzzy band until it is Endo H_f treated (right-hand gel). The sample of "delta10-15" rFH was eluted from an anion-exchange column and six peak fractions collected and run on SDS-PAGE under reducing (R) or (for four fractions) non-reducing (NR) conditions (right-hand gel), then stained with Coomassie blue.MW=molecular weight markers as indicated to left and right of the gels.

[0077] FIG. 7 is a schematic summary of a route to therapeutic versions of FH.

Example 1

Attempted Expression of Non-Codon-Optimised DNA Encoding FH

[0078] Human FH-encoding DNA was amplified from cDNA, and inserted into the yeast expression vector pPICZalphaB, and KM71 H P. pastoris cells were duly transformed. Cell colonies grew on high antibiotic-containing plates, consistent with the presence of multiple copies of the gene in the transformed cells. We failed, however, to detect (on SDS-PAGE, stained with Coomassie Blue) any evidence of FH expression in mini-scale cultures. Nor was any detectable recombinant FH produced in shaker-flask cultures. We next checked to see if protein expression by transformed cells could be detected under ideal expression conditions (as may be achieved in a one-litre fermentor in which oxygen and nutrient levels are maintained at near-optimal levels) and by using more sensitive detection methods (Western-dot-blot, see FIG. 2); notwithstanding these steps and even with the additional use of a larger-scale (three-litre) fermentation, no recombinant FH product could be detected.

[0079] In further attempts to find evidence for the expression of even small amounts of recombinant FH, a portion of the supernatant was concentrated (for Western-dot-blot) while the remainder was diluted (to reduce salt concentration) and loaded onto a HiTrap (GE Healthcare) heparin-affinity chromatography column at pH 6. A sample from a one-step elution (expected to wash all of the protein off in a small volume) with 1 M NaCl (in the equilibration buffer used for the HiTrap heparin column) was also assayed in a Western-dot-blot.

[0080] Detection was attempted using a standard Western-blotting technique with both a commercial polyclonal anti-FH antibody and secondary antibody coupled to horseradish peroxidase. With the exception of the positive controls (consisting of the primary anti-FH antibody, the secondary antibody, and human plasma-derived FH purchased from Complement Technology, Texas) no positive signal was detectable (see FIG. 2).

[0081] Thus, we demonstrated that provision of multiple-milligram, let alone multiple-gram, quantities of recombinant FH from wt FH-encoding DNA, despite the use of a heterologous expression system that is known to be particularly suitable for extracellular proteins containing disulfides and that has been used for expression of shorter segments of FH, is far from a straightforward matter.

Example 2

Development, Purification and Characterisation of Codon-Optimised Human Factor H

[0082] Codon optimisation aimed at human FH expression in P. pastoris was carried out by consultation between the inventors and Geneart (Regensburg, Germany) using their proprietary techniques and GeneOptimizer® software.

[0083] The nucleic acid sequence of a codon-optimised form of human FH, for expression in P. pastoris, is significantly different (it has 76% sequence identity) to the native DNA sequence (see FIG. 1).

[0084] The codon-optimised DNA sequence was synthesised by Geneart and then cloned into an Invitrogen-purchased P. pastoris-based expression vector, pPICZ alpha B-vector, which had been restricted using appropriate restriction enzymes.

[0085] The vector was transformed into E. coli in order to amplify the DNA, yielding several 10s of μg of plasmid DNA. This was purified, linearised (to enhance homologous recombination) and then transformed (using electroporation) into P. pastoris strain, KM71 H. Selection of P. pastoris clones containing the expression plasmid was achieved by streaking transformed yeast onto rich-media plates containing a range of concentrations of an antibiotic marker. Colonies that grew on high antibiotic-containing plates were screened for protein expression.

[0086] After filtration to remove cells, the supernatant from the fermentor was diluted one-in-five with distilled water and applied to a self-poured XK-Heparin column (Heparin FastFlow resin--from GE Healthcare). Elution was accomplished with a linear gradient, over six column volumes, from 20 mM potassium phosphate buffer (pH 6.0) to the same buffer substituted with 1 M NaCl. Fractions containing protein were pooled and the glycans were removed by incubating the sample with Endoglycosidase H-mannose binding protein fusion protein (Endo H_f, New England Biolabs) at 37° C. Protein was then applied to a Concanavalin A (GE Healthcare) column and then to mannose-binding-resin (New England Biolabs) to remove P. pastoris-derived glycans and the Endo H_f. As an alternative to Endo H_f, an exoglycosydase may be utilised so as to retain more of the glycans on the recombinant product, which might enhance solubility.

[0087] The sample was further purified on a self-poured Poros-Heparin chromatography column and eluted, over 20 column volumes, with a linear gradient from PBS to PBS plus 1 M NaCl. The final purification step involved anion exchange on a MonoQ column. The protein was eluted by a gradient, over 20 column volumes, from 20 mM glycine buffer (pH 9.5) to the same buffer supplemented with 1 M NaCl.

[0088] Exemplary results of such a purification, followed by extensive biophysical and functional characterisation and validation, are shown in FIGS. 3A-3J. The yield of protein from this procedure, that had not been optimised, was about 1.5-2.5 mg of protein from one litre.

Example 3

Further Development of Human and Mouse FH Variants Using Codon-Optimised DNA; Elaboration to Enhance Therapeutic Efficacy

[0089] In a first step, a set of 11 plasmid vectors (vector numbers 1 through 11) was designed by the inventors (FIGS. 4A and 4B) in order to further exemplify the utility and versatility of expression of a synthetic codon-optimised gene in P. pastoris. This set of vectors was designed so as to allow "cutting and pasting" of DNA encoding FH between vectors so as to maximise the number of secretion pathways that could be easily explored for each of the targeted FH variants. The aim was to produce mouse FH in addition to human FH, since mouse FH is needed for trials in mice.

[0090] In a second step, the 11 DNA inserts (see FIGS. 5A and 5B for sequence information) intended for codon optimisation were designed by the inventors based on (i) the desired amino acid residue sequences, (ii) the requirement for suitable endonuclease restriction sites, (iii) the incorporation of appropriate secretion signal sequences (peptides) at the N termini of the target proteins to promote secretion into the growth media, (iv) pursuit of the strategies summarised in FIG. 7 aimed at amassing the information required to optimise a biotherapeutic product derived from FH.

[0091] In a third step, codon optimisation and gene synthesis to create construct numbers 1 through 11 (summarised in FIGS. 5A and 5B) were carried out by Geneart (Regensburg, Germany) using their proprietary techniques and GeneOptimizer® software. Geneart were also contracted to incorporate the 11 constructs into inventor-supplied plasmids to generate vector numbers 1 through 11 (FIGS. 4A and 4B).

[0092] In the production of recombinant human (rhFH) described in Example 2 we employed a pre-pro leader (signal) sequence to direct secretion of rhFH, thereby facilitating purification. In that work, the pro-region was separated from the target sequence by an endopeptidase (kex2 protease)-cleavage site followed by two Glu-Ala dipeptides introduced to enhance cleavage-site accessibility. Native sequence generation relied upon kex2 protease to remove the pro-region, followed by dipeptidyl aminopeptidase action of the ste13-gene product to perform Glu-Ala removal. Incomplete cleavage by ste13 sometimes resulted in potentially immunogenic N-terminal Glu-Ala pairs. To eliminate this possibility, codons encoding one or both of said Glu-Ala dipeptides were avoided during creation of vector number 1 and additionally construct 1 was designed to exploit the native secretion signal sequence of hFH and processing by yeast secretion-pathway enzymes. Hence, using vector number 1 the N-terminal expression artefact (NH₂-Glu-Ala) that was included in our initial recombinant hFH is absent, and the presence of a previously present cloning artefact (Ala-Gly) is circumvented; in addition, using vector number 1, rhFH is in effect mutated to yield the protective haplotype (I62, Y402) (creating IY-hFH).

[0093] Pichia pastoris normally introduces high mannose-type N-glycans at Asn-Xaa-Thr/Ser sequons resulting in heterogenous, potentially immunogenic, products. These glycans lack terminal sialic acids and are probably susceptible to rapid clearance via hepatic asialoglycoprotein receptors. On the other hand, glycosylation may assist folding and stability of the recombinant protein and in the original study we removed P. pastoris N-glycans from rhFH enzymatically after expression and before purification or after the first purification step. Construct number 2 was designed so that Asn residues at N-glycosylation sites are replaced with Gln residues (FIGS. 5A and 5B) (to create aIIQ-IY-hFH). Thus vector number 2 allows assessment of the consequences of producing FH lacking eight normally occupied (out of nine potential) N-glycosylation sequons by mutating the relevant Asn residues to Gln residues. Thus using vector number 2 we produced, secreted (relying on the human-FH secretion signal sequence) and purified aIIQ-IY-hFH corresponding to the protective haplotype but with no N-glycosylation sites (see FIG. 6). We demonstrated that this material was glycan-free on the basis that no difference was observed in migration on SDS-PAGE before and after treatment with Endo H.

[0094] Construct 3 exploits the amber codon to allow replacement of a potentially N-glycosylated Asn residues in IY-hFH with an unnatural amino acid such as p-(propargoxy)phenylalanine (pPpa) (to create unN-IY-hFH) (see FIGS. 5A and 5B). Low long-term immunogenicity and enhanced half-life are essential properties in biotherapeutics suitable for supplementation of human FH function in patients. Attachment of poly(ethylene) glycols (PEGs) is a proven strategy in this respect (see e.g. PEGylation, successful approach to drug delivery. Veronese F M, Pasut G. Drug Discov Today. 2005; 10:1451-8). Alternatives to PEGylation include conjugation with biodegradable polysialic acid chains that may have advantages over PEGs where high and repeated doses are involved (see e.g. Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids. Gregoriadis G, Jain S, Papaioannou I, Laing P. Int J Pharm 2005 300:125-30). It will be understood that numerous other polymers could be conjugated to hFH to improve its biotherapeutic potential. Randomly placed PEGylation or polysialylation for example, on primary amines is straightforward but frequently results in a heterogenous product and steric interference with binding regions on the protein. Far more desirable is site-specific modification. We are able to exploit this desirable option thanks to our use of P. pastoris as our preferred expression system. Indeed, a very significant advantage of P. pastoris over a non-yeast eukaryotic expression system is the possibility of easily replacing one or possibly two relevant Asn residues with non-naturally encoded amino acid residues (this is possible with other eukaryotic expression systems but is less straightforward and would not be expected to produce protein in the required yields).

[0095] Thus by transfecting P. pastoris with vector number 3, along with a plasmid carrying the requisite tRNAs and aminoacyl tRNA transferase, we introduce the option of site-specific covalent modification with a chemically synthesised polymer that should mask the altered residue and eliminate a glycosylation site while potentially enhancing other biotherapeutic properties of the protein. It should be noted that these residues are not directly involved in binding to other proteins since they are normally N-glycosylated and they lie within modules of FH that we have previously shown not to be involved in C3b or GAG-binding. The system for incorporation of an unnatural amino acid used is the one developed by Schultz (Expanding the genetic repertoire of the methylotrophic meast Pichia pastoris. Young T S, Ahmad I, Brock A, Schultz P G. Biochemistry 2009 48:2643-2653) for incorporation of pPpa that is suitable for side-chain modification using "click" chemistry. This utilises an orthogonal tRNA/tRNA and aminoacyl-tRNA synthetase pair developed in E. coli using directed evolution. This allows, in the first place, the biological and biophysical properties of unN-IY-hFH to be compared to those of IY-FH (after enzymatic deglycosylation) and allQ-IY-hFH. It will be understood that another unnatural amino acid could be incorporated instead of pPpa, which would provide alternative chemical routes to conjugation; for example, we could incorporate an unnatural amino acid with an azo-group or other reactive group. Many such possibilities are discussed in the above-cited paper by Young et al the contents of which are hereby incorporated in its entirety by reference. It will also be understood that other residues besides the Asn residues in N-glycosylation sites, for example the Ser or Thr residue that is found two residues after the Asn residue, could be replaced with unnatural amino acids.

[0096] Subsequently, click chemistry is utilised to PEGylate unN-IY-hFH creating our candidate therapeutic product, PEGylated-hFH (FIG. 7); for comparison, we non-specifically PEGylate Lys residues within allQ-IY-hFH (to create PEG^x-hFH). The creation of these proteins is as follows. Azo-derivitised PEGs are available commercially and these react with the propargyl group of pPpa in a Cu(I)-catalysed azide-alkyne cycloaddition to give a high yield of the 1,2,3-triazole. It will be understood that it is possible to incorporate azo-amino acid residues instead of pPpa and then to use propargyl-PEG as a conjugate. It will also be understood that conjugations with other polymers would be equally feasible. In this way we create site-specifically PEGylated versions of FH. It is possible to explore different chain lengths, and the use of branched chains. For comparison with the products of site-specific conjugation, we use well-established protocols that randomly conjugate succinamide-ester activated PEGs to primary amines of the recombinant protein (see Peptide and protein PEGylation: a review of problems and solutions. Veronese F M. Biomaterials. 2001, 22:405-17 and references therein). Using homogenous preparations of activated PEGs at appropriate stoichiometric ratios and by fractionating and characterising the products, one obtains well-defined positional isomers of mono/di-PEGylated protein. These operations are performed on IY-hFH creating PEG''-IY-hFH. Thus it is possible to compare the relative merits of site-specific and random PEGylation. It will be understood that a similar approach may readily be extended to polysialylation instead of PEGylation.

[0097] With regard to comparisons of the various products--e.g. hFH, IY-hFH, allQ-IY-hFH, unN-IY-hFH, PEG-hFH and PEG^x-hFH--we explore their C3b- and GAG-binding properties and their bioactivities. Thus, pure and authenticated samples are tested for the following: (i) Ability to act as a cofactor for factor I-catalysed cleavage of C3b (see FIG. 3D) (Enzymic assay of C3b receptor on intact cells and solubilised cells. Sim E, Sim R B. Biochem J. 1983, 210: 567-76); (ii) Ability to promote acceleration of decay of C3bBb assembled on a surface plasmon resonance (SPR) sensor chip (Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay. Harris C L, Pettigrew D M, Lea S M, Morgan B P. J. Immunol. 2007 178:352-9)(see FIG. 3F); (iii) Affinity for C3b immobilised on a sensor chip as measured by SPR (A new map of glycosaminoglycan and C3b-binding sites on factor H. Schmidt C Q, Herbert A P, Kavanagh D, Gandy C, Fenton C J, Blaum B S, Lyon M, Uhrin D, Barlow P N. J. Immunol. 2008 181:2610-9)(see FIG. 3G; (iv) Affinity for GAGs as measured by heparin-affinity chromatography or gel-mobility shift assay (Disease-associated sequence variations congregate in a polyanion recognition patch on human factor H revealed in three-dimensional structure. Herbert A P, Uhrin D, Lyon M, Pangburn M K, Barlow P N. J Biol. Chem. 2006 281:16512-20); (v) Ability to protect sheep erythrocytes from complement-mediated haemolysis by FH-depleted human sera (available from Complement Technology)--a standard biological assay for human FH (Critical role of the C-terminal domains of factor H in regulating complement activation at cell surfaces. Ferreira V P, Herbert A P, Hocking H G, Barlow P N, Pangburn M K. J. Immunol. 2006 177:6308-16)(see FIG. 3J); (vi) Ability to protect human cells from complement-mediated injury (Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells. Barilla-LaBarca M K, Liszewski M K, Lambris J D, Hourcade D, Atkinson J P. J. Immunol. 2002 168:6298-304; Inhibiting complement activation on cells at the step of C3 cleavage. Liszewski M K, Fang C J, Atkinson J P. Vaccine. 2008, 26 Suppl 8:122-7.

[0098] In construct number 4 two amber codons have been incorporated and the protein product is suitable for site-specific placement of a pair of conjugates. With this construct it will be possible to explore the feasibility of introducing a second PEGylation site although it is expected that there may be a decrease in yield that generally accompanies each unnatural amino acid-residue incorporation. In this example, we have chosen conjugation sites on adjacent modules (modules 12 and 13) in the middle of the protein. Not only could these sites by PEGylated without compromising binding sites lying elsewhere in the FH molecule, they could be used for attachment of fluorescent probes resulting in fluorescent versions of human FH with potential applications in fluorescent microscopy and histology as well as diagnostics. Alternatively these sites could be used for conjugation with paramagnetic moieties that can be exploited in electron paramagnetic resonance spectroscopy to provide distance measurements between probes and, by inference, structural information that will help to generate hypotheses and the design of protein engineering approaches aimed at optimising FH efficacy.

[0099] Vectors 4 and 5 incorporate DNA encoding the yeast alpha-factor secretion signal peptide since it is potentially advantageous to explore secretion pathways other then the pathway that deals with the natural human FH secretion signal peptide. Vector 4 incorporates the codons for NH₂-Glu-Ala, while vector 5 does not, thereby providing opportunities to examine the role of the Glu-Ala spacer in terms of efficiency of proteolytic processing of the secretion signal peptide.

[0100] Vector 6 (utilising the alpha-factor/no-EA strategy) incorporates a construct encoding an example of a FH deletion. This term refers to versions of FH that are missing one or more central domains (or modules) within the region that connects together the two main C3b and GAG-binding sites proximal to the N and C termini. Such deletions represent an opportunity to create more compact version of hFH for research and therapeutic applications. In the current example (vector 6) modules 10-15 are deleted (for result, see FIG. 6). It will be appreciated that given the modularity of the FH structure it is possible to delete any number or combinations of modules (or to truncate FH at either end to create FH truncations). It is also facile to replace any of these deleted domains with homologous or non-homologous domains from other proteins. Vector 11 has been designed for production of an example of a FH mutant that can readily be produced in useful amounts using our strategy. In this example, nine basic amino acid residues have been replaced with Gln (neutral) residues. The basic amino acids selected in this case form a striking electropositive patch on module 13 of human FH (The central portion of factor H (modules 10-15) is compact and contains a structurally deviant CCP module. Schmidt C Q, Herbert A P, Mertens H D, Guariento M, Soares D C, Uhrin D, Rowe A J, Svergun D I, Barlow P N. J Mol. Biol. 2009 Epub. October 14.) which seems unlikely to have evolved by chance and may have an as yet unrecognised binding role in the biological mechanism of action of FH. Thus we exploit our protein production strategy both to make therapeutic proteins and to make versions of FH for assay that shed light on structure-function relationships and hence on engineering of designer versions of FH with superior therapeutic efficacy.

[0101] The subset of vectors numbered 7 through 10 were designed for production of mouse FH (mFH) in P. pastoris using codon-optimised DNA. These protein products assist in the assessment of FH as a biotherapeutic in mouse-based models of disease. The natural mFH secretion signal sequence is exploited in vectors 7, 9 and 10 while vector 8 contains DNA for the yeast alpha-factor secretion signal (no Glu-Ala). Construct 7 encodes wild-type mFH and constructs 8 and 9 encode the mouse equivalents of the allQ- and unN- (i.e. amber) versions of human FH (i.e. as in the human versions, one or two of the N-glycosylation sites of mFH are re-engineered as sites of site-specific conjugation) (allQ-mFH and unN-mFH). PEGylated (or polysialylated proteins) are constructed as described for hFH. Construct 10 encodes a two-amber-codon version of mFH in which the remaining glycosylation sites (except those in modules 1-4 and 19-20) have been substituted, Asn to Gln.

[0102] To evaluate clinical potential of the protein products of vectors 1-11, we begin with the products of vectors 7-10 and test these in (i) the FH-knockout mouse (FH.sup.-/-) that has uncontrolled plasma C3 activation and develops DDD (Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H. Pickering M C, Cook H T, Warren J, Bygrave A E, Moss J, Walport M J, Botto M. Nat Genet. 2002 31:424-8) and retinal abnormalities (Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction. Coffey P J, Gias C, McDermott C J, Lundh P, Pickering M C, Sethi C, Bird A, Fitzke F W, Maass A, Chen L L, Holder G E, Luthert P J, Salt T E, Moss S E, Greenwood J. Proc Natl Acad Sci USA. 2007 104:16651-6), and (ii) the FH transgenic mouse (CFH-/- delta16-20 (in which, effectively, the truncated FH consisting of modules 1-15 replaces full-length FH) that develops aHUS (Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains. Pickering M C, de Jorge E G, Martinez-Barricarte R, Recalde S, Garcia-Layana A, Rose K L, Moss J, Walport M J, Cook H T, de Cordoba S R, Botto M. J Exp Med 2007 204:1249-56.). We select the best candidate(s) based on a range of considerations including yield of protein, bioassays and standard toxicology studies. For example, allQ-mFH, PEG-mFH and/or PEGx-mFH (likely to have low immunogenicity) will be injected i.v./i.p. into the FH.sup.-/- mouse. Levels of complement components C3, factor B and naturally expressed mouse FH (as well as the recombinant mFH) are measured by ELISA to titrate optimal doses of mFH needed to achieve maximal complement regulation in the serum and to assess mFH half-lives. With the dosing schedule optimised we evaluate the efficacy of mFH against DDD and retinal abnormalities. Survival, renal function (urinary albumin, serum urea) and retinal abnormalities (behavioural and electrophysiological studies) of the FH.sup.-/- mice over a period of eight months (kidney)/24 months (retina) will be assessed and compared to untreated FH.sup.-/- mice. Histological studies (light microscopy, immunofluoresence and fluorescent and electron microscopy) are used to assess differences in glomerular and retinal pathology in the two groups. Any generation of antibodies against mFH in these FH-deficient mice is assessed by ELISA-based assays. The utility of our product(s) in aHUS is determined in analogous experiments in the CFH.sup.-/- delta 16-20 mouse.

[0103] We are continuing to improve the yields of hFH by further DNA manipulation and optimisation of fermentation technology, aiming to achieve production levels in the region of grams of protein per 10-litre fermentation. In the literature on P. pastoris, expression levels of 100-500 mg or more protein per litre have been reported. Numerous strategies available for the improvement of yield include: further enhancements of DNA sequence to decrease RNA secondary structure; elimination of potential proteolytic sites where possible; wider screening and selection for high copy-number transformants arising from multiple integration events; choice of culture conditions e.g. agitation, oxygen supply, pH, temperature, and addition of reagents (e.g. EDTA, amine salts, casamino acids) to minimise proteolysis; timing and rates of glycerol/methanol feeds (reviewed in for example Expression of recombinant proteins in Pichia pastoris. Li P, Anumanthan A, Gao X G, Ilangovan K, Suzara V V, Duzgune N, Renugopalakrishnan V. Appl Biochem Biotechnol. 2007 142:105-24).

Sequence CWU 1

1

1313642DNAHomo sapiens 1ggagattgca atgaacttcc tccaagaaga aatacagaaa ttctgacagg ttcctggtct 60gaccaaacat atccagaagg cacccaggct atctataaat gccgccctgg atatagatct 120cttggaaatg taataatggt atgcaggaag ggagaatggg ttgctcttaa tccattaagg 180aaatgtcaga aaaggccctg tggacatcct ggagatactc cttttggtac ttttaccctt 240acaggaggaa atgtgtttga atatggtgta aaagctgtgt atacatgtaa tgaggggtat 300caattgctag gtgagattaa ttaccgtgaa tgtgacacag atggatggac caatgatatt 360cctatatgtg aagttgtgaa gtgtttacca gtgacagcac cagagaatgg aaaaattgtc 420agtagtgcaa tggaaccaga tcgggaatac cattttggac aagcagtacg gtttgtatgt 480aactcaggct acaagattga aggagatgaa gaaatgcatt gttcagacga tggtttttgg 540agtaaagaga aaccaaagtg tgtggaaatt tcatgcaaat ccccagatgt tataaatgga 600tctcctatat ctcagaagat tatttataag gagaatgaac gatttcaata taaatgtaac 660atgggttatg aatacagtga aagaggagat gctgtatgca ctgaatctgg atggcgtccg 720ttgccttcat gtgaagaaaa atcatgtgat aatccttata ttccaaatgg tgactactca 780cctttaagga ttaaacacag aactggagat gaaatcacgt accagtgtag aaatggtttt 840tatcctgcaa cccggggaaa tacagccaaa tgcacaagta ctggctggat acctgctccg 900agatgtacct tgaaaccttg tgattatcca gacattaaac atggaggtct atatcatgag 960aatatgcgta gaccatactt tccagtagct gtaggaaaat attactccta ttactgtgat 1020gaacactttg agactccgtc aggaagttac tgggatcaca ttcattgcac acaagatgga 1080tggtcgccag cagtaccatg cctcagaaaa tgttattttc cttatttgga aaatggatat 1140aatcaaaatt atggaagaaa gtttgtacag ggtaaatcta tagacgttgc ctgccatcct 1200ggctacgctc ttccaaaagc gcagaccaca gttacatgta tggagaatgg ctggtctcct 1260actcccagat gcatccgtgt caaaacatgt tccaaatcaa gtatagatat tgagaatggg 1320tttatttctg aatctcagta tacatatgcc ttaaaagaaa aagcgaaata tcaatgcaaa 1380ctaggatatg taacagcaga tggtgaaaca tcaggatcaa ttacatgtgg gaaagatgga 1440tggtcagctc aacccacgtg cattaaatct tgtgatatcc cagtatttat gaatgccaga 1500actaaaaatg acttcacatg gtttaagctg aatgacacat tggactatga atgccatgat 1560ggttatgaaa gcaatactgg aagcaccact ggttccatag tgtgtggtta caatggttgg 1620tctgatttac ccatatgtta tgaaagagaa tgcgaacttc ctaaaataga tgtacactta 1680gttcctgatc gcaagaaaga ccagtataaa gttggagagg tgttgaaatt ctcctgcaaa 1740ccaggattta caatagttgg acctaattcc gttcagtgct accactttgg attgtctcct 1800gacctcccaa tatgtaaaga gcaagtacaa tcatgtggtc cacctcctga actcctcaat 1860gggaatgtta aggaaaaaac gaaagaagaa tatggacaca gtgaagtggt ggaatattat 1920tgcaatcctg gatttctaat gaagggacct aataaaattc aatgtgttga tggagagtgg 1980acaactttac cagtgtgtat tgtggaggag agtacctgtg gagatatacc tgaacttgaa 2040catggctggg cccagctttc ttcccctcct tattactatg gagattcagt ggaattcaat 2100tgctcagaat catttacaat gattggacac agatcaatta cgtgtattca tggagtatgg 2160acccaacttc cccagtgtgt ggcaatagat aaacttaaga agtgcaaatc atcaaattta 2220attatacttg aggaacattt aaaaaacaag aaggaattcg atcataattc taacataagg 2280tacagatgta gaggaaaaga aggatggata cacacagtct gcataaatgg aagatgggat 2340ccagaagtga actgctcaat ggcacaaata caattatgcc cacctccacc tcagattccc 2400aattctcaca atatgacaac cacactgaat tatcgggatg gagaaaaagt atctgttctt 2460tgccaagaaa attatctaat tcaggaagga gaagaaatta catgcaaaga tggaagatgg 2520cagtcaatac cactctgtgt tgaaaaaatt ccatgttcac aaccacctca gatagaacac 2580ggaaccatta attcatccag gtcttcacaa gaaagttatg cacatgggac taaattgagt 2640tatacttgtg agggtggttt caggatatct gaagaaaatg aaacaacatg ctacatggga 2700aaatggagtt ctccacctca gtgtgaaggc cttccttgta aatctccacc tgagatttct 2760catggtgttg tagctcacat gtcagacagt tatcagtatg gagaagaagt tacgtacaaa 2820tgttttgaag gttttggaat tgatgggcct gcaattgcaa aatgcttagg agaaaaatgg 2880tctcaccctc catcatgcat aaaaacagat tgtctcagtt tacctagctt tgaaaatgcc 2940atacccatgg gagagaagaa ggatgtgtat aaggcgggtg agcaagtgac ttacacttgt 3000gcaacatatt acaaaatgga tggagccagt aatgtaacat gcattaatag cagatggaca 3060ggaaggccaa catgcagaga cacctcctgt gtgaatccgc ccacagtaca aaatgcttat 3120atagtgtcga gacagatgag taaatatcca tctggtgaga gagtacgtta tcaatgtagg 3180agcccttatg aaatgtttgg ggatgaagaa gtgatgtgtt taaatggaaa ctggacggaa 3240ccacctcaat gcaaagattc tacaggaaaa tgtgggcccc ctccacctat tgacaatggg 3300gacattactt cattcccgtt gtcagtatat gctccagctt catcagttga gtaccaatgc 3360cagaacttgt atcaacttga gggtaacaag cgaataacat gtagaaatgg acaatggtca 3420gaaccaccaa aatgcttaca tccgtgtgta atatcccgag aaattatgga aaattataac 3480atagcattaa ggtggacagc caaacagaag ctttattcga gaacaggtga atcagttgaa 3540tttgtgtgta aacggggata tcgtctttca tcacgttctc acacattgcg aacaacatgt 3600tgggatggga aactggagta tccaacttgt gcaaaaagat ag 364223645DNAArtificialCodon Optimised sequence for P. pastoris expression of human factor H 2gaggattgta acgagttgcc accaagaaga aacactgaga tcttgactgg ttcttggagt 60gatcaaactt acccagaggg tactcaggct atctacaagt gtagaccagg ttacagatcc 120ttgggtaacg ttatcatggt ttgtagaaag ggtgagtggg ttgcattgaa cccattgaga 180aagtgtcaga aaagaccatg tggtcaccca ggtgatactc cattcggtac tttcactttg 240actggtggta acgttttcga gtacggtgtt aaggctgttt acacttgtaa cgagggttac 300cagttgttgg gagagatcaa ctacagagag tgtgatactg acggatggac taacgacatt 360ccaatctgtg aagttgttaa gtgtttgcca gttactgctc cagagaacgg aaagattgtt 420tcctccgcta tggaaccaga tagagagtac cacttcggac aggctgttag attcgtttgt 480aactccggtt acaagattga aggtgacgaa gagatgcact gttctgatga cggtttctgg 540tccaaagaaa agccaaagtg tgttgagatc tcctgtaagt ccccagacgt tattaacggt 600tccccaatct cccaaaagat catctacaaa gagaacgaga gattccagta caagtgtaac 660atgggttacg agtactctga aagaggtgac gctgtttgta ctgaatctgg atggagacca 720ttgccatcct gtgaagagaa gtcctgtgac aacccataca ttccaaacgg tgactactcc 780ccattgagaa tcaagcacag aactggtgac gagatcactt accagtgtag aaatggtttc 840tacccagcta ctagaggtaa cactgctaag tgtacttcca ctggatggat tccagctcca 900agatgtactt tgaagccatg tgactaccca gatatcaagc acggtggttt gtaccacgag 960aacatgagaa ggccatactt cccagttgct gttggaaagt actactccta ctactgtgac 1020gaacacttcg aaactccatc tggttcttac tgggaccaca tccactgtac tcaagatggt 1080tggtccccag ctgttccatg tttgagaaaa tgttacttcc catacttgga gaacggttac 1140aaccagaact acggtagaaa gttcgttcag ggaaagtcca ttgacgttgc ttgtcatcca 1200ggttacgctt tgccaaaggc tcagactact gttacttgta tggaaaacgg ttggtcccct 1260actcctagat gtatcagagt taagacttgt tccaagtcct ccatcgacat tgagaacggt 1320ttcatttccg agtcccagta cacttacgct ttgaaagaga aggctaagta ccagtgtaaa 1380ttgggatacg ttactgctga cggtgaaact tccggatcaa tcacatgtgg aaaagacgga 1440tggagtgctc aaccaacttg tatcaagtct tgtgacatcc cagttttcat gaacgctaga 1500actaagaacg acttcacatg gttcaagttg aacgacactt tggactacga atgtcacgac 1560ggttacgaat ctaacactgg ttccactact ggttccatcg tttgtggtta caatggatgg 1620agtgacttgc caatctgtta cgagagagag tgcgagttgc caaagatcga cgttcatttg 1680gttccagaca gaaagaagga ccagtacaaa gttggagagg ttttgaagtt ctcctgtaag 1740ccaggtttca ctatcgttgg tccaaactcc gttcagtgtt accacttcgg tttgtctcca 1800gacttgccta tctgtaaaga gcaggttcaa tcctgcggac caccaccaga attgttgaac 1860ggtaacgtta aagaaaagac taaagaagag tacggtcact ccgaagttgt tgagtactac 1920tgtaacccaa gattcttgat gaagggtcca aacaagatcc aatgtgttga cggtgagtgg 1980actactttgc cagtttgtat cgttgaagag tccacttgtg gtgacattcc agaattggaa 2040cacggatggg ctcaattgtc atccccacca tactactacg gtgactccgt tgaattcaac 2100tgttccgagt ccttcactat gattggtcac agatccatca catgtatcca cggtgtttgg 2160actcaattgc cacagtgtgt tgctatcgac aagttgaaga agtgtaaatc atccaacctt 2220atcatcttgg aggaacactt gaagaacaag aaagagttcg accacaactc caacatcaga 2280tacagatgta gaggtaaaga gggatggatc cacactgttt gtatcaacgg tagatgggac 2340cctgaagtta actgttccat ggctcagatt cagttgtgtc caccaccacc acaaattcca 2400aactcccaca acatgactac tactttgaac tacagagatg gtgaaaaggt ttccgttttg 2460tgtcaagaga actacttgat ccaagagggt gaagagatca catgtaagga cggtagatgg 2520cagtccatcc ctttgtgtgt tgagaagatc ccatgttccc aaccacctca aattgagcac 2580ggtactatca actcttccag atcctctcaa gagtcttacg ctcacggtac taagttgtcc 2640tacacttgtg agggaggttt cagaatctct gaggaaaacg agactacttg ttacatggga 2700aagtggtcat ctccaccaca atgtgaagga ttgccttgta agtctccacc agagatttct 2760cacggtgttg ttgctcacat gtccgactct taccaatacg gagaagaggt tacctacaag 2820tgtttcgagg gtttcggtat tgatggtcca gctatcgcta agtgtttggg agaaaagtgg 2880tcccatcctc catcctgtat caagactgat tgtttgtcct tgccatcctt cgaaaacgct 2940atcccaatgg gagaaaagaa ggacgtttac aaggctggtg aacaagttac ttatacttgt 3000gctacttact acaagatgga cggtgcttcc aacgttactt gtatcaactc cagatggact 3060ggtagaccaa cttgtagaga cacttcctgt gttaacccac caactgttca gaacgcttac 3120atcgtttcca gacagatgtc taagtaccca tccggagaac gtgttagata ccaatgtaga 3180tccccatacg agatgttcgg tgacgaagag gttatgtgtt tgaacggtaa ttggactgaa 3240ccaccacagt gtaaggactc cactggtaag tgtggtccac ctccaccaat tgacaacggt 3300gacatcactt ctttcccttt gtccgtttac gctccagctt cttccgttga gtaccagtgt 3360cagaacttgt accagttgga gggtaacaag agaatcactt gtagaaacgg acaatggagt 3420gagccaccaa agtgtttgca cccatgtgtt atctccagag aaatcatgga aaactacaac 3480attgctttga gatggactgc taaacagaag ttgtactcca gaactggtga atccgttgag 3540ttcgtttgta agagaggtta cagattgtcc tccagatccc acactttgag aactacatgt 3600tgggacggaa aattggagta cccaacttgt gctaagagat agtag 364533733DNAArtificialCodon optimised human hactor H variant 3ggcgcgccgg atccaaaaat gagattgttg gctaagatca tctgtttgat gttgtgggct 60atctgtgttg ctgaggactg taacgaattg ccaccgcgga gaaacactga gattttgact 120ggttcctggt ccgatcaaac ttacccagag ggtactcagg ctatctacaa gtgtagacca 180ggttacagat ccttgggtaa catcatcatg gtttgtagaa agggtgagtg ggttgctttg 240aacccattga gaaagtgtca gaaaagacca tgtggtcacc caggtgatac tccattcggt 300actttcactt tgactggtgg taacgttttc gagtacggtg ttaaggctgt ttacacttgt 360aacgagggtt accagttgtt gggtgagatc aactacagag agtgtgatac tgacggttgg 420actaacgaca ttccaatctg tgaggttgtt aagtgtttgc cagttactgc tccagagaac 480ggtaagattg tttcctccgc tatggaacca gatagagagt accacttcgg tcaggctgtt 540agattcgttt gtaactccgg ttacaagatt gaaggtgacg aagagatgca ctgttctgat 600gacggtttct ggtccaaaga aaagccaaag tgtgttgaga tttcctgtaa gtccccagac 660gttattaacg gttccccaat ctcccaaaag atcatctaca aagagaacga gagattccag 720tacaagtgta acatgggtta cgagtactct gaaagaggtg acgctgtttg tactgaatct 780ggttggagac cattgccatc ctgtgaagag aagtcctgtg acaacccata cattccaaac 840ggtgactact ccccattgag aatcaagcac agaactggtg acgagatcac ttaccagtgt 900agaaacggtt tctacccagc tactagaggt aacactgcta agtgtacttc cactggttgg 960attccagctc caagatgtac tttgaagcca tgtgactacc cagatatcaa gcacggtggt 1020ttgtaccacg agaacatgag aagaccatac ttcccagttg ctgttggaaa gtactactcc 1080tactactgtg acgaacactt cgaaactcca tctggttctt actgggacca catccactgt 1140actcaagatg gttggtcccc agctgttcca tgtttgagaa aatgttactt cccatacttg 1200gagaacggtt acaaccagaa ctacggtaga aagttcgttc agggaaagtc cattgacgtt 1260gcttgtcatc caggttacgc tttgccaaag gctcagacta ctgttacttg tatggaaaac 1320ggttggtccc ctactcctag atgtatcaga gttaagactt gttccaagtc ctccatcgac 1380attgagaacg gtttcatttc cgagtcccag tacacttacg ctttgaaaga gaaggctaag 1440taccagtgta aattgggata cgttactgct gacggtgaaa cttccggttc catcacttgt 1500ggtaaggatg gttggtctgc tcaaccaact tgtatcaagt cttgtgacat cccagttttc 1560atgaacgcta gaactaagaa cgacttcaca tggttcaagt tgaacgacac tttggactac 1620gaatgtcacg acggttacga atctaacact ggttccacta ctggttccat cgtttgtggt 1680tacaacggtt ggtctgactt gccaatctgt tacgagagag agtgcgagtt gccaaagatc 1740gacgttcatt tggttccaga cagaaagaag gaccagtaca aggttggtga ggttttgaag 1800ttctcctgta agccaggttt cactatcgtt ggtccaaact ccgttcagtg ttaccatttc 1860ggtttgtccc cagacttgcc tatttgtaaa gagcaggttc agtcttgcgg tccaccacca 1920gaattgttga acggtaacgt taaagaaaag actaaagaag agtacggtca ctctgaggtt 1980gttgagtact actgtaaccc aagattcttg atgaagggtc caaacaagat ccaatgtgtt 2040gacggtgagt ggactacttt gccagtttgt atcgttgaag agtccacttg tggtgacatt 2100ccagaattgg aacacggttg ggctcaattg tcatccccac catactacta cggtgactcc 2160gttgagttca actgttccga gtccttcact atgattggtc acagatccat cacatgtatc 2220cacggtgttt ggactcaatt gccacagtgt gttgctatcg acaagttgaa gaagtgtaaa 2280tcctccaact tgatcatctt ggaggaacac ttgaagaaca agaaagagtt cgaccacaac 2340tccaacatca gatacagatg tagaggtaaa gagggttgga ttcacactgt ttgtatcaac 2400ggtagatggg accctgaagt taactgttcc atggctcaga ttcagttgtg tccaccacct 2460ccacaaattc caaactccca caacatgact actactttga actacagaga tggtgagaag 2520gtttccgttt tgtgtcaaga gaactacttg atccaagagg gtgaggaaat cacttgtaag 2580gacggtagat ggcaatccat cccattgtgt gttgagaaga tcccatgttc ccaaccacca 2640caaattgagc acggtactat caactcttcc agatcctctc aagagtctta cgctcacggt 2700actaagttgt cctacacttg tgagggtggt ttcagaatct ctgaggaaaa cgagactact 2760tgttacatgg gaaagtggtc ctctccacca caatgtgaag gtttgccttg taagtctcca 2820ccagagattt ctcacggtgt tgttgctcac atgtccgact cttaccaata cggtgaagag 2880gttacttaca agtgtttcga gggtttcggt attgatggtc cagctatcgc taagtgtttg 2940ggtgaaaagt ggtcccatcc tccatcctgt atcaagactg actgtttgtc cttgccatct 3000ttcgagaacg ctatcccaat gggtgaaaag aaggacgttt acaaggctgg tgaacaggtt 3060acatacactt gtgctactta ctacaagatg gacggtgctt ccaacgttac ttgtatcaac 3120tccagatgga ctggtagacc aacttgtaga gacacttcct gtgttaaccc accaactgtt 3180cagaacgctt acatcgtttc cagacagatg tctaagtacc catccggtga gagagttaga 3240taccaatgta gatccccata cgagatgttc ggtgacgaag aggttatgtg tttgaacggt 3300aattggactg aaccaccaca gtgtaaggac tccactggta agtgtggtcc acctccacca 3360attgacaacg gtgacatcac ttctttccca ttgtccgttt acgctccagc ttcttccgtt 3420gagtaccagt gtcagaactt gtaccagttg gagggtaaca agagaatcac ttgtagaaac 3480ggacaatggt ctgagccacc aaagtgtttg cacccatgtg ttatctccag agaaatcatg 3540gaaaactaca acattgcttt gagatggact gctaagcaga agttgtactc cagaacaggt 3600gagtctgttg agtttgtttg taagagaggt tacagattgt cctccagatc ccacactttg 3660agaactacat gttgggacgg aaagttggag tacccaactt gtgctaagag ataatgagcg 3720gccgcttaat taa 373343733DNAArtificialCodon optimised human hactor H variant 4ggcgcgccgg atccaaaaat gagattgttg gctaagatca tctgtttgat gttgtgggct 60atctgtgttg ctgaggactg taacgaattg ccaccgcgga gaaacactga gattttgact 120ggttcctggt ccgatcaaac ttacccagag ggtactcagg ctatctacaa gtgtagacca 180ggttacagat ccttgggtaa catcatcatg gtttgtagaa agggtgagtg ggttgctttg 240aacccattga gaaagtgtca gaaaagacca tgtggtcacc caggtgatac tccattcggt 300actttcactt tgactggtgg taacgttttc gagtacggtg ttaaggctgt ttacacttgt 360aacgagggtt accagttgtt gggtgagatc aactacagag agtgtgatac tgacggttgg 420actaacgaca ttccaatctg tgaggttgtt aagtgtttgc cagttactgc tccagagaac 480ggtaagattg tttcctccgc tatggaacca gatagagagt accacttcgg tcaggctgtt 540agattcgttt gtaactccgg ttacaagatt gaaggtgacg aagagatgca ctgttctgat 600gacggtttct ggtccaaaga aaagccaaag tgtgttgaga tttcctgtaa gtccccagac 660gttattaacg gttccccaat ctcccaaaag atcatctaca aagagaacga gagattccag 720tacaagtgta acatgggtta cgagtactct gaaagaggtg acgctgtttg tactgaatct 780ggttggagac cattgccatc ctgtgaagag aagtcctgtg acaacccata cattccaaac 840ggtgactact ccccattgag aatcaagcac agaactggtg acgagatcac ttaccagtgt 900agaaacggtt tctacccagc tactagaggt aacactgcta agtgtacttc cactggttgg 960attccagctc caagatgtac tttgaagcca tgtgactacc cagatatcaa gcacggtggt 1020ttgtaccacg agaacatgag aagaccatac ttcccagttg ctgttggaaa gtactactcc 1080tactactgtg acgaacactt cgaaactcca tctggttctt actgggacca catccactgt 1140actcaagatg gttggtcccc agctgttcca tgtttgagaa aatgttactt cccatacttg 1200gagaacggtt acaaccagaa ctacggtaga aagttcgttc agggaaagtc cattgacgtt 1260gcttgtcatc caggttacgc tttgccaaag gctcagacta ctgttacttg tatggaaaac 1320ggttggtccc ctactcctag atgtatcaga gttaagactt gttccaagtc ctccatcgac 1380attgagaacg gtttcatttc cgagtcccag tacacttacg ctttgaaaga gaaggctaag 1440taccagtgta aattgggata cgttactgct gacggtgaaa cttccggttc catcacttgt 1500ggtaaggatg gttggtctgc tcaaccaact tgtatcaagt cttgtgacat cccagttttc 1560atgaacgcta gaactaagaa cgacttcaca tggttcaagt tgcaagacac tttggactac 1620gaatgtcacg acggttacga atctaacact ggttccacta ctggttccat cgtttgtggt 1680tacaacggtt ggtctgactt gccaatctgt tacgagagag agtgcgagtt gccaaagatc 1740gacgttcatt tggttccaga cagaaagaag gaccagtaca aggttggtga ggttttgaag 1800ttctcctgta agccaggttt cactatcgtt ggtccaaact ccgttcagtg ttaccatttc 1860ggtttgtccc cagacttgcc tatttgtaaa gagcaggttc agtcttgcgg tccaccacca 1920gaattgttga acggtaacgt taaagaaaag actaaagaag agtacggtca ctctgaggtt 1980gttgagtact actgtaaccc aagattcttg atgaagggtc caaacaagat ccaatgtgtt 2040gacggtgagt ggactacttt gccagtttgt atcgttgaag agtccacttg tggtgacatt 2100ccagaattgg aacacggttg ggctcaattg tcatccccac catactacta cggtgactcc 2160gttgagttcc aatgttccga gtccttcact atgattggtc acagatccat cacatgtatc 2220cacggtgttt ggactcaatt gccacagtgt gttgctatcg acaagttgaa gaagtgtaaa 2280tcctccaact tgatcatctt ggaggaacac ttgaagaaca agaaagagtt cgaccacaac 2340tccaacatca gatacagatg tagaggtaaa gagggttgga ttcacactgt ttgtatcaac 2400ggtagatggg accctgaagt tcaatgttcc atggctcaga ttcagttgtg tccaccacct 2460ccacaaattc caaactccca ccaaatgact actactttga actacagaga tggtgagaag 2520gtttccgttt tgtgtcaaga gaactacttg atccaagagg gtgaggaaat cacttgtaag 2580gacggtagat ggcaatccat cccattgtgt gttgagaaga tcccatgttc ccaaccacca 2640caaattgagc acggtactat ccaatcttcc agatcctctc aagagtctta cgctcacggt 2700actaagttgt cctacacttg tgagggtggt ttcagaatct ctgaggaaca agagactact 2760tgttacatgg gaaagtggtc ctctccacca caatgtgaag gtttgccttg taagtctcca 2820ccagagattt ctcacggtgt tgttgctcac atgtccgact cttaccaata cggtgaagag 2880gttacttaca agtgtttcga gggtttcggt attgatggtc cagctatcgc taagtgtttg 2940ggtgaaaagt ggtcccatcc tccatcctgt atcaagactg actgtttgtc cttgccatct 3000ttcgagaacg ctatcccaat gggtgaaaag aaggacgttt acaaggctgg tgaacaggtt 3060acatacactt gtgctactta ctacaagatg gacggtgctt cccaagttac ttgtatcaac 3120tccagatgga ctggtagacc aacttgtaga gacacttcct gtgttaaccc accaactgtt 3180cagaacgctt acatcgtttc cagacagatg tctaagtacc catccggtga gagagttaga 3240taccaatgta gatccccata cgagatgttc ggtgacgaag aggttatgtg tttgaacggt 3300caatggactg aaccaccaca gtgtaaggac tccactggta agtgtggtcc acctccacca 3360attgacaacg gtgacatcac ttctttccca ttgtccgttt acgctccagc ttcttccgtt 3420gagtaccagt gtcagaactt gtaccagttg gagggtaaca agagaatcac ttgtagaaac 3480ggacaatggt ctgagccacc aaagtgtttg cacccatgtg ttatctccag agaaatcatg 3540gaaaactaca acattgcttt gagatggact gctaagcaga agttgtactc cagaacaggt 3600gagtctgttg agtttgtttg taagagaggt tacagattgt cctccagatc ccacactttg 3660agaactacat gttgggacgg aaagttggag tacccaactt gtgctaagag ataatgagcg 3720gccgcttaat taa

373353733DNAArtificialCodon optimised human hactor H variant 5ggcgcgccgg atccaaaaat gagattgttg gctaagatca tctgtttgat gttgtgggct 60atctgtgttg ctgaggactg taacgaattg ccaccgcgga gaaacactga gattttgact 120ggttcctggt ccgatcaaac ttacccagag ggtactcagg ctatctacaa gtgtagacca 180ggttacagat ccttgggtaa catcatcatg gtttgtagaa agggtgagtg ggttgctttg 240aacccattga gaaagtgtca gaaaagacca tgtggtcacc caggtgatac tccattcggt 300actttcactt tgactggtgg taacgttttc gagtacggtg ttaaggctgt ttacacttgt 360aacgagggtt accagttgtt gggtgagatc aactacagag agtgtgatac tgacggttgg 420actaacgaca ttccaatctg tgaggttgtt aagtgtttgc cagttactgc tccagagaac 480ggtaagattg tttcctccgc tatggaacca gatagagagt accacttcgg tcaggctgtt 540agattcgttt gtaactccgg ttacaagatt gaaggtgacg aagagatgca ctgttctgat 600gacggtttct ggtccaaaga aaagccaaag tgtgttgaga tttcctgtaa gtccccagac 660gttattaacg gttccccaat ctcccaaaag atcatctaca aagagaacga gagattccag 720tacaagtgta acatgggtta cgagtactct gaaagaggtg acgctgtttg tactgaatct 780ggttggagac cattgccatc ctgtgaagag aagtcctgtg acaacccata cattccaaac 840ggtgactact ccccattgag aatcaagcac agaactggtg acgagatcac ttaccagtgt 900agaaacggtt tctacccagc tactagaggt aacactgcta agtgtacttc cactggttgg 960attccagctc caagatgtac tttgaagcca tgtgactacc cagatatcaa gcacggtggt 1020ttgtaccacg agaacatgag aagaccatac ttcccagttg ctgttggaaa gtactactcc 1080tactactgtg acgaacactt cgaaactcca tctggttctt actgggacca catccactgt 1140actcaagatg gttggtcccc agctgttcca tgtttgagaa aatgttactt cccatacttg 1200gagaacggtt acaaccagaa ctacggtaga aagttcgttc agggaaagtc cattgacgtt 1260gcttgtcatc caggttacgc tttgccaaag gctcagacta ctgttacttg tatggaaaac 1320ggttggtccc ctactcctag atgtatcaga gttaagactt gttccaagtc ctccatcgac 1380attgagaacg gtttcatttc cgagtcccag tacacttacg ctttgaaaga gaaggctaag 1440taccagtgta aattgggata cgttactgct gacggtgaaa cttccggttc catcacttgt 1500ggtaaggatg gttggtctgc tcaaccaact tgtatcaagt cttgtgacat cccagttttc 1560atgaacgcta gaactaagaa cgacttcaca tggttcaagt tgcaagacac tttggactac 1620gaatgtcacg acggttacga atctaacact ggttccacta ctggttccat cgtttgtggt 1680tacaacggtt ggtctgactt gccaatctgt tacgagagag agtgcgagtt gccaaagatc 1740gacgttcatt tggttccaga cagaaagaag gaccagtaca aggttggtga ggttttgaag 1800ttctcctgta agccaggttt cactatcgtt ggtccaaact ccgttcagtg ttaccatttc 1860ggtttgtccc cagacttgcc tatttgtaaa gagcaggttc agtcttgcgg tccaccacca 1920gaattgttga acggtaacgt taaagaaaag actaaagaag agtacggtca ctctgaggtt 1980gttgagtact actgtaaccc aagattcttg atgaagggtc caaacaagat ccaatgtgtt 2040gacggtgagt ggactacttt gccagtttgt atcgttgaag agtccacttg tggtgacatt 2100ccagaattgg aacacggttg ggctcaattg tcatccccac catactacta cggtgactcc 2160gttgagttcc aatgttccga gtccttcact atgattggtc acagatccat cacatgtatc 2220cacggtgttt ggactcaatt gccacagtgt gttgctatcg acaagttgaa gaagtgtaaa 2280tcctccaact tgatcatctt ggaggaacac ttgaagaaca agaaagagtt cgaccacaac 2340tccaacatca gatacagatg tagaggtaaa gagggttgga ttcacactgt ttgtatcaac 2400ggtagatggg accctgaagt tcaatgttcc atggctcaga ttcagttgtg tccaccacct 2460ccacaaattc caaactccca ccaaatgact actactttga actacagaga tggtgagaag 2520gtttccgttt tgtgtcaaga gaactacttg atccaagagg gtgaggaaat cacttgtaag 2580gacggtagat ggcaatccat cccattgtgt gttgagaaga tcccatgttc ccaaccacca 2640caaattgagc acggtactat ccaatctagt agatcctctc aagagtctta cgctcacggt 2700actaagttgt cctacacttg tgagggtggt ttcagaatct ctgaggaata ggagactact 2760tgttacatgg gaaagtggtc ctctccacca caatgtgaag gtttgccttg taagtctcca 2820ccagagattt ctcacggtgt tgttgctcac atgtccgact cttaccaata cggtgaagag 2880gttacttaca agtgtttcga gggtttcggt attgatggtc cagctatcgc taagtgtttg 2940ggtgaaaagt ggtcccatcc tccatcctgt atcaagactg actgtttgtc cttgccatct 3000ttcgagaacg ctatcccaat gggtgaaaag aaggacgttt acaaggctgg tgaacaggtt 3060acatacactt gtgctactta ctacaagatg gacggtgctt cccaagttac ttgtatcaac 3120tccagatgga ctggtagacc aacttgtaga gacacttcct gtgttaaccc accaactgtt 3180cagaacgctt acatcgtttc cagacagatg tctaagtacc catccggtga gagagttaga 3240taccaatgta gatccccata cgagatgttc ggtgacgaag aggttatgtg tttgaacggt 3300caatggactg aaccaccaca gtgtaaggac tccactggta agtgtggtcc acctccacca 3360attgacaacg gtgacatcac ttctttccca ttgtccgttt acgctccagc ttcttccgtt 3420gagtaccagt gtcagaactt gtaccagttg gagggtaaca agagaatcac ttgtagaaac 3480ggacaatggt ctgagccacc aaagtgtttg cacccatgtg ttatctccag agaaatcatg 3540gaaaactaca acattgcttt gagatggact gctaagcaga agttgtactc cagaacaggt 3600gagtctgttg agtttgtttg taagagaggt tacagattgt cctccagatc ccacactttg 3660agaactacat gttgggacgg aaagttggag tacccaactt gtgctaagag ataatgagcg 3720gccgcttaat taa 373363676DNAArtificialCodon optimised human hactor H variant 6ggcgcgcctg caggtgagga ctgtaacgaa ttgccaccgc ggagaaacac tgagattttg 60actggttcct ggtccgatca aacttaccca gagggtactc aggctatcta caagtgtaga 120ccaggttaca gatccttggg taacatcatc atggtttgta gaaagggtga gtgggttgct 180ttgaacccat tgagaaagtg tcagaaaaga ccatgtggtc acccaggtga tactccattc 240ggtactttca ctttgactgg tggtaacgtt ttcgagtacg gtgttaaggc tgtttacact 300tgtaacgagg gttaccagtt gttgggtgag atcaactaca gagagtgtga tactgacggt 360tggactaacg acattccaat ctgtgaggtt gttaagtgtt tgccagttac tgctccagag 420aacggtaaga ttgtttcctc cgctatggaa ccagatagag agtaccactt cggtcaggct 480gttagattcg tttgtaactc cggttacaag attgaaggtg acgaagagat gcactgttct 540gatgacggtt tctggtccaa agaaaagcca aagtgtgttg agatttcctg taagtcccca 600gacgttatta acggttcccc aatctcccaa aagatcatct acaaagagaa cgagagattc 660cagtacaagt gtaacatggg ttacgagtac tctgaaagag gtgacgctgt ttgtactgaa 720tctggttgga gaccattgcc atcctgtgaa gagaagtcct gtgacaaccc atacattcca 780aacggtgact actccccatt gagaatcaag cacagaactg gtgacgagat cacttaccag 840tgtagaaacg gtttctaccc agctactaga ggtaacactg ctaagtgtac ttccactggt 900tggattccag ctccaagatg tactttgaag ccatgtgact acccagatat caagcacggt 960ggtttgtacc acgagaacat gagaagacca tacttcccag ttgctgttgg aaagtactac 1020tcctactact gtgacgaaca cttcgaaact ccatctggtt cttactggga ccacatccac 1080tgtactcaag atggttggtc cccagctgtt ccatgtttga gaaaatgtta cttcccatac 1140ttggagaacg gttacaacca gaactacggt agaaagttcg ttcagggaaa gtccattgac 1200gttgcttgtc atccaggtta cgctttgcca aaggctcaga ctactgttac ttgtatggaa 1260aacggttggt cccctactcc tagatgtatc agagttaaga cttgttccaa gtcctccatc 1320gacattgaga acggtttcat ttccgagtcc cagtacactt acgctttgaa agagaaggct 1380aagtaccagt gtaaattggg atacgttact gctgacggtg aaacttccgg ttccatcact 1440tgtggtaagg atggttggtc tgctcaacca acttgtatca agtcttgtga catcccagtt 1500ttcatgaacg ctagaactaa gaacgacttc acatggttca agttgaacga cactttggac 1560tacgaatgtc acgacggtta cgaatctaac actggttcca ctactggttc catcgtttgt 1620ggttacaacg gttggtctga cttgccaatc tgttacgaga gagagtgcga gttgccaaag 1680atcgacgttc atttggttcc agacagaaag aaggaccagt acaaggttgg tgaggttttg 1740aagttctcct gtaagccagg tttcactatc gttggtccaa actccgttca gtgttaccat 1800ttcggtttgt ccccagactt gcctatttgt aaagagcagg ttcagtcttg cggtccacca 1860ccagaattgt tgaacggtaa cgttaaagaa aagactaaag aagagtacgg tcactctgag 1920gttgttgagt actactgtaa cccaagattc ttgatgaagg gtccaaacaa gatccaatgt 1980gttgacggtg agtggactac tttgccagtt tgtatcgttg aagagtccac ttgtggtgac 2040attccagaat tggaacacgg ttgggctcaa ttgtcatccc caccatacta ctacggtgac 2100tccgttgagt tctagtgttc cgagtccttc actatgattg gtcacagatc catcacatgt 2160atccacggtg tttggactca attgccacag tgtgttgcta tcgacaagtt gaagaagtgt 2220aaatcctcca acttgatcat cttggaggaa cacttgaaga acaagaaaga gttcgaccac 2280aactccaaca tcagatacag atgtagaggt aaagagggtt ggattcacac tgtttgtatc 2340aacggtagat gggaccctga agttaactgt tccatggctc agattcagtt gtgtccacca 2400cctccacaaa ttccaaactc ccacaacatg actactactt tgaactacag agatggtgag 2460aaggtttccg ttttgtgtca agagaactac ttgatccaag agggtgagga aatcacttgt 2520aaggacggta gatggcaatc catcccattg tgtgttgaga agatcccatg ttcccaacca 2580ccacaaattg agcacggtac tatcaactct tccagatcct ctcaagagtc ttacgctcac 2640ggtactaagt tgtcctacac ttgtgagggt ggtttcagaa tctctgagga ataggagact 2700acttgttaca tgggaaagtg gtcctctcca ccacaatgtg aaggtttgcc ttgtaagtct 2760ccaccagaga tttctcacgg tgttgttgct cacatgtccg actcttacca atacggtgaa 2820gaggttactt acaagtgttt cgagggtttc ggtattgatg gtccagctat cgctaagtgt 2880ttgggtgaaa agtggtccca tcctccatcc tgtatcaaga ctgactgttt gtccttgcca 2940tctttcgaga acgctatccc aatgggtgaa aagaaggacg tttacaaggc tggtgaacag 3000gttacataca cttgtgctac ttactacaag atggacggtg cttccaacgt tacttgtatc 3060aactccagat ggactggtag accaacttgt agagacactt cctgtgttaa cccaccaact 3120gttcagaacg cttacatcgt ttccagacag atgtctaagt acccatccgg tgagagagtt 3180agataccaat gtagatcccc atacgagatg ttcggtgacg aagaggttat gtgtttgaac 3240ggtaattgga ctgaaccacc acagtgtaag gactccactg gtaagtgtgg tccacctcca 3300ccaattgaca acggtgacat cacttctttc ccattgtccg tttacgctcc agcttcttcc 3360gttgagtacc agtgtcagaa cttgtaccag ttggagggta acaagagaat cacttgtaga 3420aacggacaat ggtctgagcc accaaagtgt ttgcacccat gtgttatctc cagagaaatc 3480atggaaaact acaacattgc tttgagatgg actgctaagc agaagttgta ctccagaaca 3540ggtgagtctg ttgagtttgt ttgtaagaga ggttacagat tgtcctccag atcccacact 3600ttgagaacta catgttggga cggaaagttg gagtacccaa cttgtgctaa gagataatga 3660gcggccgctt aattaa 367673934DNAArtificialCodon optimised human hactor H variant 7ggcgcgccgg atccaaaaat gagattccca tccatcttca ctgctgtttt gttcgctgct 60tcttctgctt tggctgctcc agttaacact actactgagg acgagactgc tcaaattcca 120gctgaggctg ttattggtta ctctgacttg gaaggtgatt tcgacgttgc tgttttgcca 180ttctccaact ccactaacaa cggtttgttg ttcatcaaca ctactatcgc ttccattgct 240gctaaagaag agggagtttc cctcgagaag agagaggact gtaacgaatt gccaccgcgg 300agaaacactg agattttgac tggttcctgg tccgatcaaa cttacccaga gggtactcag 360gctatctaca agtgtagacc aggttacaga tccttgggta acgttatcat ggtttgtaga 420aagggtgagt gggttgcttt gaacccattg agaaagtgtc agaaaagacc atgtggtcac 480ccaggtgata ctccattcgg tactttcact ttgactggtg gtaacgtttt cgagtacggt 540gttaaggctg tttacacttg taacgagggt taccagttgt tgggtgagat caactacaga 600gagtgtgata ctgacggttg gactaacgac attccaatct gtgaggttgt taagtgtttg 660ccagttactg ctccagagaa cggtaagatt gtttcctccg ctatggaacc agatagagag 720taccacttcg gtcaggctgt tagattcgtt tgtaactccg gttacaagat tgaaggtgac 780gaagagatgc actgttctga tgacggtttc tggtccaaag aaaagccaaa gtgtgttgag 840atttcctgta agtccccaga cgttattaac ggttccccaa tctcccaaaa gatcatctac 900aaagagaacg agagattcca gtacaagtgt aacatgggtt acgagtactc tgaaagaggt 960gacgctgttt gtactgaatc tggttggaga ccattgccat cctgtgaaga gaagtcctgt 1020gacaacccat acattccaaa cggtgactac tccccattga gaatcaagca cagaactggt 1080gacgagatca cttaccagtg tagaaacggt ttctacccag ctactagagg taacactgct 1140aagtgtactt ccactggttg gattccagct ccaagatgta ctttgaagcc atgtgactac 1200ccagatatca agcacggtgg tttgtaccac gagaacatga gaagaccata cttcccagtt 1260gctgttggaa agtactactc ctactactgt gacgaacact tcgaaactcc atctggttct 1320tactgggacc acatccactg tactcaagat ggttggtccc cagctgttcc atgtttgaga 1380aaatgttact tcccatactt ggagaacggt tacaaccaga accatggtag aaagttcgtt 1440cagggaaagt ccattgacgt tgcttgtcat ccaggttacg ctttgccaaa ggctcagact 1500actgttactt gtatggaaaa cggttggtcc cctactccta gatgtatcag agttaagact 1560tgttccaagt cctccatcga cattgagaac ggtttcattt ccgagtccca gtacacttac 1620gctttgaaag agaaggctaa gtaccagtgt aaattgggat acgttactgc tgacggtgaa 1680acttccggtt ccatcacttg tggtaaggat ggttggtctg ctcaaccaac ttgtatcaag 1740tcttgtgaca tcccagtttt catgaacgct agaactaaga acgacttcac atggttcaag 1800ttgaacgaca ctttggacta cgaatgtcac gacggttacg aatctaacac tggttccact 1860actggttcca tcgtttgtgg ttacaacggt tggtctgact tgccaatctg ttacgagaga 1920gagtgcgagt tgccaaagat cgacgttcat ttggttccag acagaaagaa ggaccagtac 1980aaggttggtg aggttttgaa gttctcctgt aagccaggtt tcactatcgt tggtccaaac 2040tccgttcagt gttaccattt cggtttgtcc ccagacttgc ctatttgtaa agagcaggtt 2100cagtcttgcg gtccaccacc agaattgttg aacggtaacg ttaaagaaaa gactaaagaa 2160gagtacggtc actctgaggt tgttgagtac tactgtaacc caagattctt gatgaagggt 2220ccaaacaaga tccaatgtgt tgacggtgag tggactactt tgccagtttg tatcgttgaa 2280gagtccactt gtggtgacat tccagaattg gaacacggtt gggctcaatt gtcatcccca 2340ccatactact acggtgactc cgttgagttc aactgttccg agtccttcac tatgattggt 2400cacagatcca tcacatgtat ccacggtgtt tggactcaat tgccacagtg tgttgctatc 2460gacaagttga agaagtgtaa atcctccaac ttgatcatct tggaggaaca cttgaagaac 2520aagaaagagt tcgaccacaa ctccaacatc agatacagat gtagaggtaa agagggttgg 2580attcacactg tttgtatcaa cggtagatgg gaccctgaag ttaactgttc catggctcag 2640attcagttgt gtccaccacc tccacaaatt ccaaactccc acaacatgac tactactttg 2700aactacagag atggtgagaa ggtttccgtt ttgtgtcaag agaactactt gatccaagag 2760ggtgaggaaa tcacttgtaa ggacggtaga tggcaatcca tcccattgtg tgttgagaag 2820atcccatgtt cccaaccacc acaaattgag cacggtacta tcaactcttc cagatcctct 2880caagagtctt acgctcacgg tactaagttg tcctacactt gtgagggtgg tttcagaatc 2940tctgaggaaa acgagactac ttgttacatg ggaaagtggt cctctccacc acaatgtgaa 3000ggtttgcctt gtaagtctcc accagagatt tctcacggtg ttgttgctca catgtccgac 3060tcttaccaat acggtgaaga ggttacttac aagtgtttcg agggtttcgg tattgatggt 3120ccagctatcg ctaagtgttt gggtgaaaag tggtcccatc ctccatcctg tatcaagact 3180gactgtttgt ccttgccatc tttcgagaac gctatcccaa tgggtgaaaa gaaggacgtt 3240tacaaggctg gtgaacaggt tacatacact tgtgctactt actacaagat ggacggtgct 3300tccaacgtta cttgtatcaa ctccagatgg actggtagac caacttgtag agacacttcc 3360tgtgttaacc caccaactgt tcagaacgct tacatcgttt ccagacagat gtctaagtac 3420ccatccggtg agagagttag ataccaatgt agatccccat acgagatgtt cggtgacgaa 3480gaggttatgt gtttgaacgg taattggact gaaccaccac agtgtaagga ctccactggt 3540aagtgtggtc cacctccacc aattgacaac ggtgacatca cttctttccc attgtccgtt 3600tacgctccag cttcttccgt tgagtaccag tgtcagaact tgtaccagtt ggagggtaac 3660aagagaatca cttgtagaaa cggacaatgg tctgagccac caaagtgttt gcacccatgt 3720gttatctcca gagaaatcat ggaaaactac aacattgctt tgagatggac tgctaagcag 3780aagttgtact ccagaacagg tgagtctgtt gagtttgttt gtaagagagg ttacagattg 3840tcctccagat cccacacttt gagaactaca tgttgggacg gaaagttgga gtacccaact 3900tgtgctaaga gataatgagc ggccgcttaa ttaa 393482848DNAArtificialCodon optimised human hactor H variant 8ggcgcgccgg atccaaaaat gagattccca tccatcttca ctgctgtttt gttcgctgct 60tcttctgctt tggctgctcc agttaacact actactgagg acgagactgc tcaaattcca 120gctgaggctg ttattggtta ctctgacttg gaaggtgatt tcgacgttgc tgttttgcca 180ttctccaact ccactaacaa cggtttgttg ttcatcaaca ctactatcgc ttccattgct 240gctaaagaag agggagtttc cctcgagaag agagaggact gtaacgaatt gccaccgcgg 300agaaacactg agattttgac tggttcctgg tccgatcaaa cttacccaga gggtactcag 360gctatctaca agtgtagacc aggttacaga tccttgggta acattatcat ggtttgtaga 420aagggtgagt gggttgcttt gaacccattg agaaagtgtc agaaaagacc atgtggtcac 480ccaggtgata ctccattcgg tactttcact ttgactggtg gtaacgtttt cgagtacggt 540gttaaggctg tttacacttg taacgagggt taccagttgt tgggtgagat caactacaga 600gagtgtgata ctgacggttg gactaacgac attccaatct gtgaggttgt taagtgtttg 660ccagttactg ctccagagaa cggtaagatt gtttcctccg ctatggaacc agatagagag 720taccacttcg gtcaggctgt tagattcgtt tgtaactccg gttacaagat tgaaggtgac 780gaagagatgc actgttctga tgacggtttc tggtccaaag aaaagccaaa gtgtgttgag 840atttcctgta agtccccaga cgttattaac ggttccccaa tctcccaaaa gatcatctac 900aaagagaacg agagattcca gtacaagtgt aacatgggtt acgagtactc tgaaagaggt 960gacgctgttt gtactgaatc tggttggaga ccattgccat cctgtgaaga gaagtcctgt 1020gacaacccat acattccaaa cggtgactac tccccattga gaatcaagca cagaactggt 1080gacgagatca cttaccagtg tagaaacggt ttctacccag ctactagagg taacactgct 1140aagtgtactt ccactggttg gattccagct ccaagatgta ctttgaagcc atgtgactac 1200ccagatatca agcacggtgg tttgtaccac gagaacatga gaagaccata cttcccagtt 1260gctgttggaa agtactactc ctactactgt gacgaacact tcgaaactcc atctggttct 1320tactgggacc acatccactg tactcaagat ggttggtccc cagctgttcc atgtttgaga 1380aaatgttact tcccatactt ggagaacggt tacaaccaga actacggtag aaagttcgtt 1440cagggaaagt ccattgacgt tgcttgtcat ccaggttacg ctttgccaaa ggctcagact 1500actgttactt gtatggaaaa cggttggtcc cctactccta gatgtatcag agttaagact 1560tgttccaagt cctccatcga cattgagaac ggtttcattt ccgagtccca gtacacttac 1620gctttgaaag agaaggctaa gtaccagtgt aaattgggat acgttactgc tgacggtgaa 1680acttccggtt ccatcacttg tggtaaggat ggttggtctg ctcaaccaac ttgtatcaag 1740tcttgtgaca tcccagtttt catgaacgct agaactaaga acgacttcac atggttcaag 1800ttgaacgaca ctttggacta cgaatgtcac gacggttacg aatctaacac tggttccact 1860actggttcca tcgtttgtgg ttacaacggt tggtctgact tgccaatctg ttacgagttg 1920ccttgtaagt ctccaccaga gatttctcac ggtgttgttg ctcacatgtc cgactcttac 1980caatacggtg aagaggttac ttacaagtgt ttcgagggtt tcggtattga tggtccagct 2040atcgctaagt gtttgggtga aaagtggtcc catcctccat cctgtatcaa gactgactgt 2100ttgtccttgc catctttcga gaacgctatc ccaatgggtg aaaagaagga cgtttacaag 2160gctggtgaac aggttacata cacttgtgct acttactaca agatggacgg tgcttccaac 2220gttacttgta tcaactccag atggactggt agaccaactt gtagagacac ttcctgtgtt 2280aacccaccaa ctgttcagaa cgcttacatc gtttccagac agatgtctaa gtacccatcc 2340ggtgagagag ttagatacca atgtagatcc ccatacgaga tgttcggtga cgaagaggtt 2400atgtgtttga acggtaattg gactgaacca ccacagtgta aggactccac tggtaagtgt 2460ggtccacctc caccaattga caacggtgac atcacttctt tcccattgtc cgtttacgct 2520ccagcttctt ccgttgagta ccagtgtcag aacttgtacc agttggaggg taacaagaga 2580atcacttgta gaaacggaca atggtctgag ccaccaaagt gtttgcaccc atgtgttatc 2640tccagagaaa tcatggaaaa ctacaacatt gctttgagat ggactgctaa gcagaagttg 2700tactccagaa caggtgagtc tgttgagttt gtttgtaaga gaggttacag attgtcctcc 2760agatcccaca ctttgagaac tacatgttgg gacggaaagt tggagtaccc aacttgtgct 2820aagagataat gagcggccgc ttaattaa 284893742DNAArtificialCodon optimised mouse hactor H variant 9ggcgcgccgg atccaaaaat gagattgtcc gctagaatca tctggttgat cttgtggact 60gtttgtgctg ctgaggattg taaaggtcca ccaccgcggg aaaactccga gattttgtct 120ggttcttggt ccgaacaatt gtacccagag ggtactcaag ctacttacaa gtgtagacca 180ggttacagaa ctttgggtac tatcgttaag gtttgtaaga acggaaagtg ggttgcttct 240aacccatcca gaatctgtag aaagaaacca tgtggtcacc caggtgatac tccattcggt 300tccttcagat tggctgttgg ttcccaattc gagttcggtg ctaaggttgt ttacacttgt 360gacgacggtt accaattgtt gggtgagatc gactacagag aatgtggtgc tgacggttgg 420attaacgaca

tcccattgtg tgaggttgtt aagtgtttgc cagttactga gttggagaac 480ggtagaattg tttctggtgc tgctgaaact gaccaagagt actacttcgg acaggttgtt 540agattcgagt gtaactccgg tttcaagatc gaaggtcaca aagagattca ctgttccgag 600aacggtttgt ggtctaacga gaagccaaga tgtgttgaga ttttgtgtac tccaccaaga 660gttgaaaacg gtgacggtat caacgttaag ccagtttaca aagagaacga gagataccac 720tacaagtgta agcacggtta cgttccaaaa gaaagaggtg acgctgtttg tactggttct 780ggttggtcct ctcaaccatt ctgtgaagag aagagatgtt ccccaccata catcttgaac 840ggtatctaca ctccacacag aatcattcac agatccgacg acgagattag atacgaatgt 900aactacggat tctacccagt tactggttcc actgtttcca agtgtactcc aactggttgg 960attccagttc caagatgtac tttgaagcca tgtgagttcc cacaattcaa gtacggtaga 1020ttgtactacg aagagtcctt gagaccaaac ttcccagttt ccatcggtaa caagtactcc 1080tacaagtgtg acaacggttt ctctccacca tctggttact cttgggacta cttgagatgt 1140actgctcaag gttgggaacc agaggttcca tgtgttagaa agtgtgtttt ccactacgtt 1200gagaacggtg attctgctta ctgggagaag gtttacgttc aaggtcagtc cttgaaggtt 1260cagtgttaca acggttactc cttgcaaaac ggtcaggaca ctatgacttg tactgagaac 1320ggttggtcac caccaccaaa gtgtatcaga atcaagactt gttccgcttc cgacattcac 1380atcgacaacg gattcttgtc tgagtcctcc tccatttacg ctttgaacag agagacttcc 1440tacagatgta agcagggata cgttacaaac actggtgaga tttccggttc catcacttgt 1500ttgcagaatg gttggtcccc acagccatct tgtattaagt cctgtgacat gccagttttc 1560gagaactcca tcactaagaa cactagaaca tggttcaagt tgaacgacaa gttggactac 1620gagtgtttgg ttggtttcga gaacgagtac aagcacacta agggttccat cacatgtact 1680tactacggtt ggtctgacac tccatcctgt tacgaaagag agtgttccgt tccaactttg 1740gacagaaagt tggttgtttc cccaagaaaa gagaagtaca gagttggaga cttgttggag 1800ttctcttgtc actctggtca tagagttggt ccagactccg ttcaatgtta ccactttgga 1860tggtccccag gttttccaac ttgtaagggt caggttgctt cttgtgctcc accattggag 1920attttgaacg gtgagatcaa cggtgctaag aaggttgaat actcccacgg tgaagttgtt 1980aagtacgact gtaagccaag attcttgttg aagggtccaa acaagatcca atgtgttgac 2040ggtaactgga ctactttgcc agtttgtatc gaggaagaaa gaacttgcgg agacatccca 2100gaattggaac acggttccgc taagtgttct gttccaccat accaccatgg tgattccgtt 2160gagttcatct gtgaggaaaa cttcactatg atcggtcacg gttccgtttc ttgtatttcc 2220ggtaagtgga ctcagttgcc aaagtgtgtt gctactgacc agttggagaa gtgtagagtt 2280ttgaagtcca ctggtatcga ggctatcaag ccaaagttga ctgagttcac tcacaactcc 2340actatggact acaaatgtag agacaagcaa gagtacgaga gatccatctg tatcaacggt 2400aaatgggacc cagaaccaaa ctgtacttcc aagacttctt gtccaccacc accacaaatt 2460ccaaacactc aggttatcga gactactgtt aagtacttgg acggtgagaa gttgtccgtt 2520ttgtgtcagg acaactactt gactcaagac tccgaagaga tggtttgtaa ggacggtaga 2580tggcaatctt tgccaagatg tatcgagaag atcccatgtt ctcagccacc aactattgag 2640cacggttcca ttaacttgcc aagatcctcc gaagaaagaa gagactccat cgaatcctct 2700tctcacgaac acggtactac tttctcttac gtttgtgatg acggtttcag aatcccagaa 2760gagaacagaa tcacttgtta catgggaaag tggtccactc cacctagatg tgttggtttg 2820ccatgtggtc caccaccttc tattccattg ggtactgttt ctttggagtt ggagtcctac 2880caacacggtg aagaggttac ttaccactgt tccactggtt tcggtattga tggtccagct 2940ttcattatct gtgagggtgg taagtggtct gatccaccta agtgtattaa gactgactgt 3000gacgttttgc caactgttaa gaacgctatc atcagaggta agtccaagaa gtcctacaga 3060actggagagc aggttacttt cagatgtcag tccccatacc aaatgaacgg ttccgacact 3120gttacttgtg ttaactccag atggatcggt caaccagttt gtaaggataa ctcctgtgtt 3180gatccaccac atgttccaaa cgctactatc gttactagaa ctaagaacaa gtacttgcat 3240ggtgacagag ttagatatga gtgtaacaag ccattggagt tgttcggtca agttgaggtt 3300atgtgtgaga acggtatctg gactgagaag ccaaagtgta gagactccac tggtaagtgt 3360ggtcctccac caccaattga caacggtgac atcacttctt tgtccttgcc agtttacgaa 3420cctttgtcct ccgttgagta ccaatgtcag aagtactact tgttgaaagg taagaaaact 3480atcacttgta ctaatggtaa atggtccgag ccaccaactt gtttgcacgc ttgtgttatc 3540ccagagaaca tcatggaatc ccacaacatc atcttgaagt ggagacacac tgagaagatt 3600tactctcact ccggtgagga cattgagttc ggttgtaagt acggttacta caaggctaga 3660gactctccac cattcagaac taagtgtatc aacggaacta tcaactaccc aacttgtgtt 3720taatgagcgg ccgcttaatt aa 3742103943DNAArtificialCodon optimised mouse hactor H variant 10ggcgcgccgg atccaaaaat gagattccca tccatcttca ctgctgtttt gttcgctgct 60tcttctgctt tggctgctcc agttaacact actactgagg acgagactgc tcaaattcca 120gctgaggctg ttattggtta ctctgacttg gaaggtgatt tcgacgttgc tgttttgcca 180ttctccaact ccactaacaa cggtttgttg ttcatcaaca ctactatcgc ttccattgct 240gctaaagaag agggagtttc cctcgagaag agagaggatt gtaaaggtcc accaccgcgg 300gaaaactccg agattttgtc tggttcttgg tccgaacaat tgtacccaga gggtactcaa 360gctacttaca agtgtagacc aggttacaga actttgggta ctatcgttaa ggtttgtaag 420aacggaaagt gggttgcttc tcaaccatcc agaatctgta gaaagaaacc atgtggtcac 480ccaggtgata ctccattcgg ttccttcaga ttggctgttg gttcccaatt cgagttcggt 540gctaaggttg tttacacttg tgacgacggt taccaattgt tgggtgagat cgactacaga 600gaatgtggtg ctgacggttg gattaacgac atcccattgt gtgaggttgt taagtgtttg 660ccagttactg agttggagaa cggtagaatt gtttctggtg ctgctgaaac tgaccaagag 720tactacttcg gacaggttgt tagattcgag tgtaactccg gtttcaagat cgaaggtcac 780aaagagattc actgttccga gaacggtttg tggtctaacg agaagccaag atgtgttgag 840attttgtgta ctccaccaag agttgaaaac ggtgacggta tcaacgttaa gccagtttac 900aaagagaacg agagatacca ctacaagtgt aagcacggtt acgttccaaa agaaagaggt 960gacgctgttt gtactggttc tggttggtcc tctcaaccat tctgtgaaga gaagagatgt 1020tccccaccat acatcttgaa cggtatctac actccacaca gaatcattca cagatccgac 1080gacgagatta gatacgaatg taactacgga ttctacccag ttactggttc cactgtttcc 1140aagtgtactc caactggttg gattccagtt ccaagatgta ctttgaagcc atgtgagttc 1200ccacaattca agtacggtag attgtactac gaagagtcct tgagaccaaa cttcccagtt 1260tccatcggta acaagtactc ctacaagtgt gacaacggtt tctctccacc atctggttac 1320tcttgggact acttgagatg tactgctcaa ggttgggaac cagaggttcc atgtgttaga 1380aagtgtgttt tccactacgt tgagaacggt gattctgctt actgggagaa ggtttacgtt 1440caaggtcagt ccttgaaggt tcagtgttac aacggttact ccttgcaaaa cggtcaggac 1500actatgactt gtactgagaa cggttggtca ccaccaccaa agtgtatcag aatcaagact 1560tgttccgctt ccgacattca catcgacaac ggattcttgt ctgagtcctc ctccatttac 1620gctttgaaca gagagacttc ctacagatgt aagcagggat acgttacaaa cactggtgag 1680atttccggtt ccatcacttg tttgcagaat ggttggtccc cacagccatc ttgtattaag 1740tcctgtgaca tgccagtttt cgagaactcc atcactaaga acactagaac atggttcaag 1800ttgaacgaca agttggacta cgagtgtttg gttggtttcg agaacgagta caagcacact 1860aagggttcca tcacatgtac ttactacggt tggtctgaca ctccatcctg ttacgaaaga 1920gagtgttccg ttccaacttt ggacagaaag ttggttgttt ccccaagaaa agagaagtac 1980agagttggag acttgttgga gttctcttgt cactctggtc atagagttgg tccagactcc 2040gttcaatgtt accactttgg atggtcccca ggttttccaa cttgtaaggg tcaggttgct 2100tcttgtgctc caccattgga gattttgaac ggtgagatca acggtgctaa gaaggttgaa 2160tactcccacg gtgaagttgt taagtacgac tgtaagccaa gattcttgtt gaagggtcca 2220aacaagatcc aatgtgttga cggtcaatgg actactttgc cagtttgtat cgaggaagaa 2280agaacttgcg gagacatccc agaattggaa cacggttccg ctaagtgttc tgttccacca 2340taccaccatg gtgattccgt tgagttcatc tgtgaggaac aattcactat gatcggtcac 2400ggttccgttt cttgtatttc cggtaagtgg actcagttgc caaagtgtgt tgctactgac 2460cagttggaga agtgtagagt tttgaagtcc actggtatcg aggctatcaa gccaaagttg 2520actgagttca ctcaccagtc cactatggac tacaaatgta gagacaagca agagtacgag 2580agatccatct gtatcaacgg taaatgggac ccagaaccac aatgtacttc caagacttct 2640tgtccaccac caccacaaat tccaaacact caggttatcg agactactgt taagtacttg 2700gacggtgaga agttgtccgt tttgtgtcag gacaactact tgactcaaga ctccgaagag 2760atggtttgta aggacggtag atggcaatct ttgccaagat gtatcgagaa gatcccatgt 2820tctcagccac caactattga gcacggttcc attaacttgc caagatcctc cgaagaaaga 2880agagactcca tcgaatcctc ttctcacgaa cacggtacta ctttctctta cgtttgtgat 2940gacggtttca gaatcccaga agagaacaga atcacttgtt acatgggaaa gtggtccact 3000ccacctagat gtgttggttt gccatgtggt ccaccacctt ctattccatt gggtactgtt 3060tctttggagt tggagtccta ccaacacggt gaagaggtta cttaccactg ttccactggt 3120ttcggtattg atggtccagc tttcattatc tgtgagggtg gtaagtggtc tgatccacct 3180aagtgtatta agactgactg tgacgttttg ccaactgtta agaacgctat catcagaggt 3240aagtccaaga agtcctacag aactggagag caggttactt tcagatgtca gtccccatac 3300caaatgcaag gttccgacac tgttacttgt gttaactcca gatggatcgg tcaaccagtt 3360tgtaaggata actcctgtgt tgatccacca catgttccac aagctactat cgttactaga 3420actaagaaca agtacttgca tggtgacaga gttagatatg agtgtaacaa gccattggag 3480ttgttcggtc aagttgaggt tatgtgtgag aacggtatct ggactgagaa gccaaagtgt 3540agagactcca ctggtaagtg tggtcctcca ccaccaattg acaacggtga catcacttct 3600ttgtccttgc cagtttacga acctttgtcc tccgttgagt accaatgtca gaagtactac 3660ttgttgaaag gtaagaaaac tatcacttgt actaatggta aatggtccga gccaccaact 3720tgtttgcacg cttgtgttat cccagagaac atcatggaat cccacaacat catcttgaag 3780tggagacaca ctgagaagat ttactctcac tccggtgagg acattgagtt cggttgtaag 3840tacggttact acaaggctag agactctcca ccattcagaa ctaagtgtat ccaaggaact 3900atcaactacc caacttgtgt ttaatgagcg gccgcttaat taa 3943113742DNAArtificialCodon optimised mouse hactor H variant 11ggcgcgccgg atccaaaaat gagattgtcc gctagaatca tctggttgat cttgtggact 60gtttgtgctg ctgaggattg taaaggtcca ccaccgcggg aaaactccga gattttgtct 120ggttcttggt ccgaacaatt gtacccagag ggtactcaag ctacttacaa gtgtagacca 180ggttacagaa ctttgggtac tatcgttaag gtttgtaaga acggaaagtg ggttgcttct 240caaccatcca gaatctgtag aaagaaacca tgtggtcacc caggtgatac tccattcggt 300tccttcagat tggctgttgg ttcccaattc gagttcggtg ctaaggttgt ttacacttgt 360gacgacggtt accaattgtt gggtgagatc gactacagag aatgtggtgc tgacggttgg 420attaacgaca tcccattgtg tgaggttgtt aagtgtttgc cagttactga gttggagaac 480ggtagaattg tttctggtgc tgctgaaact gaccaagagt actacttcgg acaggttgtt 540agattcgagt gtaactccgg tttcaagatc gaaggtcaca aagagattca ctgttccgag 600aacggtttgt ggtctaacga gaagccaaga tgtgttgaga ttttgtgtac tccaccaaga 660gttgaaaacg gtgacggtat caacgttaag ccagtttaca aagagaacga gagataccac 720tacaagtgta agcacggtta cgttccaaaa gaaagaggtg acgctgtttg tactggttct 780ggttggtcct ctcaaccatt ctgtgaagag aagagatgtt ccccaccata catcttgaac 840ggtatctaca ctccacacag aatcattcac agatccgacg acgagattag atacgaatgt 900aactacggat tctacccagt tactggttcc actgtttcca agtgtactcc aactggttgg 960attccagttc caagatgtac tttgaagcca tgtgagttcc cacaattcaa gtacggtaga 1020ttgtactacg aagagtcctt gagaccaaac ttcccagttt ccatcggtaa caagtactcc 1080tacaagtgtg acaacggttt ctctccacca tctggttact cttgggacta cttgagatgt 1140actgctcaag gttgggaacc agaggttcca tgtgttagaa agtgtgtttt ccactacgtt 1200gagaacggtg attctgctta ctgggagaag gtttacgttc aaggtcagtc cttgaaggtt 1260cagtgttaca acggttactc cttgcaaaac ggtcaggaca ctatgacttg tactgagaac 1320ggttggtcac caccaccaaa gtgtatcaga atcaagactt gttccgcttc cgacattcac 1380atcgacaacg gattcttgtc tgagtcctcc tccatttacg ctttgaacag agagacttcc 1440tacagatgta agcagggata cgttacaaac actggtgaga tttccggttc catcacttgt 1500ttgcagaatg gttggtcccc acagccatct tgtattaagt cctgtgacat gccagttttc 1560gagaactcca tcactaagaa cactagaaca tggttcaagt tgaacgacaa gttggactac 1620gagtgtttgg ttggtttcga gaacgagtac aagcacacta agggttccat cacatgtact 1680tactacggtt ggtctgacac tccatcctgt tacgaaagag agtgttccgt tccaactttg 1740gacagaaagt tggttgtttc cccaagaaaa gagaagtaca gagttggaga cttgttggag 1800ttctcttgtc actctggtca tagagttggt ccagactccg ttcaatgtta ccactttgga 1860tggtccccag gttttccaac ttgtaagggt caggttgctt cttgtgctcc accattggag 1920attttgaacg gtgagatcaa cggtgctaag aaggttgaat actcccacgg tgaagttgtt 1980aagtacgact gtaagccaag attcttgttg aagggtccaa acaagatcca atgtgttgac 2040ggtcaatgga ctactttgcc agtttgtatc gaggaagaaa gaacttgcgg agacatccca 2100gaattggaac acggttccgc taagtgttct gttccaccat accaccatgg tgattccgtt 2160gagttcatct gtgaggagta gttcactatg atcggtcacg gttccgtttc ttgtatttcc 2220ggtaagtgga ctcagttgcc aaagtgtgtt gctactgacc agttggagaa gtgtagagtt 2280ttgaagtcca ctggtatcga ggctatcaag ccaaagttga ctgagttcac tcaccagtct 2340actatggact acaaatgtag agacaagcaa gagtacgaga gatccatctg tatcaacggt 2400aaatgggacc cagaaccaca atgtacttcc aagacttctt gtccaccacc accacaaatt 2460ccaaacactc aggttatcga gactactgtt aagtacttgg acggtgagaa gttgtccgtt 2520ttgtgtcagg acaactactt gactcaagac tccgaagaga tggtttgtaa ggacggtaga 2580tggcaatctt tgccaagatg tatcgagaag atcccatgtt ctcagccacc aactattgag 2640cacggttcca ttaacttgcc aagatcctcc gaagaaagaa gagactccat cgaatcctct 2700tctcacgaac acggtactac tttctcttac gtttgtgatg acggtttcag aatcccagaa 2760gagaacagaa tcacttgtta catgggaaag tggtccactc cacctagatg tgttggtttg 2820ccatgtggtc caccaccttc tattccattg ggtactgttt ctttggagtt ggagtcctac 2880caacacggtg aagaggttac ttaccactgt tccactggtt tcggtattga tggtccagct 2940ttcattatct gtgagggtgg taagtggtct gatccaccta agtgtattaa gactgactgt 3000gacgttttgc caactgttaa gaacgctatc atcagaggta agtccaagaa gtcctacaga 3060actggagagc aggttacttt cagatgtcag tccccatacc aaatgcaagg ttccgacact 3120gttacttgtg ttaactccag atggatcggt caaccagttt gtaaggataa ctcctgtgtt 3180gatccaccac atgttccaca agctactatc gttactagaa ctaagaacaa gtacttgcat 3240ggtgacagag ttagatatga gtgtaacaag ccattggagt tgttcggtca agttgaggtt 3300atgtgtgaga acggtatctg gactgagaag ccaaagtgta gagactccac tggtaagtgt 3360ggtcctccac caccaattga caacggtgac atcacttctt tgtccttgcc agtttacgaa 3420cctttgtcct ccgttgagta ccaatgtcag aagtactact tgttgaaagg taagaaaact 3480atcacttgta ctaatggtaa atggtccgag ccaccaactt gtttgcacgc ttgtgttatc 3540ccagagaaca tcatggaatc ccacaacatc atcttgaagt ggagacacac tgagaagatt 3600tactctcact ccggtgagga cattgagttc ggttgtaagt acggttacta caaggctaga 3660gactctccac cattcagaac taagtgtatc caaggaacta tcaactaccc aacttgtgtt 3720taatgagcgg ccgcttaatt aa 3742123742DNAArtificialCodon optimised mouse hactor H variant 12ggcgcgccgg atccaaaaat gagattgtcc gctagaatca tctggttgat cttgtggact 60gtttgtgctg ctgaggattg taaaggtcca ccaccgcggg aaaactccga gattttgtct 120ggttcttggt ccgaacaatt gtacccagag ggtactcaag ctacttacaa gtgtagacca 180ggttacagaa ctttgggtac tatcgttaag gtttgtaaga acggaaagtg ggttgcttct 240caaccatcca gaatctgtag aaagaaacca tgtggtcacc caggtgatac tccattcggt 300tccttcagat tggctgttgg ttcccaattc gagttcggtg ctaaggttgt ttacacttgt 360gacgacggtt accaattgtt gggtgagatc gactacagag aatgtggtgc tgacggttgg 420attaacgaca tcccattgtg tgaggttgtt aagtgtttgc cagttactga gttggagaac 480ggtagaattg tttctggtgc tgctgaaact gaccaagagt actacttcgg acaggttgtt 540agattcgagt gtaactccgg tttcaagatc gaaggtcaca aagagattca ctgttccgag 600aacggtttgt ggtctaacga gaagccaaga tgtgttgaga ttttgtgtac tccaccaaga 660gttgaaaacg gtgacggtat caacgttaag ccagtttaca aagagaacga gagataccac 720tacaagtgta agcacggtta cgttccaaaa gaaagaggtg acgctgtttg tactggttct 780ggttggtcct ctcaaccatt ctgtgaagag aagagatgtt ccccaccata catcttgaac 840ggtatctaca ctccacacag aatcattcac agatccgacg acgagattag atacgaatgt 900aactacggat tctacccagt tactggttcc actgtttcca agtgtactcc aactggttgg 960attccagttc caagatgtac tttgaagcca tgtgagttcc cacaattcaa gtacggtaga 1020ttgtactacg aagagtcctt gagaccaaac ttcccagttt ccatcggtaa caagtactcc 1080tacaagtgtg acaacggttt ctctccacca tctggttact cttgggacta cttgagatgt 1140actgctcaag gttgggaacc agaggttcca tgtgttagaa agtgtgtttt ccactacgtt 1200gagaacggtg attctgctta ctgggagaag gtttacgttc aaggtcagtc cttgaaggtt 1260cagtgttaca acggttactc cttgcaaaac ggtcaggaca ctatgacttg tactgagaac 1320ggttggtcac caccaccaaa gtgtatcaga atcaagactt gttccgcttc cgacattcac 1380atcgacaacg gattcttgtc tgagtcctcc tccatttacg ctttgaacag agagacttcc 1440tacagatgta agcagggata cgttacaaac actggtgaga tttccggttc catcacttgt 1500ttgcagaatg gttggtcccc acagccatct tgtattaagt cctgtgacat gccagttttc 1560gagaactcca tcactaagaa cactagaaca tggttcaagt tgaacgacaa gttggactac 1620gagtgtttgg ttggtttcga gaacgagtac aagcacacta agggttccat cacatgtact 1680tactacggtt ggtctgacac tccatcctgt tacgaaagag agtgttccgt tccaactttg 1740gacagaaagt tggttgtttc cccaagaaaa gagaagtaca gagttggaga cttgttggag 1800ttctcttgtc actctggtca tagagttggt ccagactccg ttcaatgtta ccactttgga 1860tggtccccag gttttccaac ttgtaagggt caggttgctt cttgtgctcc accattggag 1920attttgaacg gtgagatcaa cggtgctaag aaggttgaat actcccacgg tgaagttgtt 1980aagtacgact gtaagccaag attcttgttg aagggtccaa acaagatcca atgtgttgac 2040ggtcaatgga ctactttgcc agtttgtatc gaggaagaaa gaacttgcgg agacatccca 2100gaattggaac acggttccgc taagtgttct gttccaccat accaccatgg tgattccgtt 2160gagttcatct gtgaggagta gttcactatg atcggtcacg gttccgtttc ttgtatttcc 2220ggtaagtgga ctcagttgcc aaagtgtgtt gctactgacc agttggagaa gtgtagagtt 2280ttgaagtcca ctggtatcga ggctatcaag ccaaagttga ctgagttcac tcaccagtcc 2340actatggact acaaatgtag agacaagcaa gagtacgaga gatccatctg tatcaacggt 2400aaatgggacc cagaaccaca atgtacttcc aagacttctt gtccaccacc accacaaatt 2460ccaaacactc aggttatcga gactactgtt aagtacttgg acggtgagaa gttgtccgtt 2520ttgtgtcagg acaactactt gactcaagac tccgaagaga tggtttgtaa ggacggtaga 2580tggcaatctt tgccaagatg tatcgagaag atcccatgtt ctcagccacc aactattgag 2640cacggttcca ttaacttgcc aagatcctcc gaagaaagaa gagactccat cgaatcctct 2700tctcacgaac acggtactac tttctcttac gtttgtgatg acggtttcag aatcccagaa 2760gagaacagaa tcacttgtta catgggaaag tggtccactc cacctagatg tgttggtttg 2820ccatgtggtc caccaccttc tattccattg ggtactgttt ctttggagtt ggagtcctac 2880caacacggtg aagaggttac ttaccactgt tccactggtt tcggtattga tggtccagct 2940ttcattatct gtgagggtgg taagtggtct gatccaccta agtgtattaa gactgactgt 3000gacgttttgc caactgttaa gaacgctatc atcagaggta agtccaagaa gtcctacaga 3060actggagagc aggttacttt cagatgtcag tccccatacc aaatgtaggg ttccgacact 3120gttacttgtg ttaactccag atggatcggt caaccagttt gtaaggataa ctcctgtgtt 3180gatccaccac atgttccaca agctactatc gttactagaa ctaagaacaa gtacttgcat 3240ggtgacagag ttagatatga gtgtaacaag ccattggagt tgttcggtca agttgaggtt 3300atgtgtgaga acggtatctg gactgagaag ccaaagtgta gagactccac tggtaagtgt 3360ggtcctccac caccaattga caacggtgac atcacttctt tgtccttgcc agtttacgaa 3420cctttgtcct ccgttgagta ccaatgtcag aagtactact tgttgaaagg taagaaaact 3480atcacttgta ctaatggtaa atggtccgag ccaccaactt gtttgcacgc ttgtgttatc 3540ccagagaaca tcatggaatc ccacaacatc atcttgaagt ggagacacac tgagaagatt 3600tactctcact ccggtgagga cattgagttc ggttgtaagt acggttacta caaggctaga 3660gactctccac cattcagaac taagtgtatc caaggaacta tcaactaccc aacttgtgtt 3720taatgagcgg ccgcttaatt aa

3742133733DNAArtificialCodon optimised human hactor H variant 13ggcgcgccgg atccaaaaat gagattgttg gctaagatca tctgtttgat gttgtgggct 60atctgtgttg ctgaggactg taacgaattg ccaccgcgga gaaacactga gattttgact 120ggttcctggt ccgatcaaac ttacccagag ggtactcagg ctatctacaa gtgtagacca 180ggttacagat ccttgggtaa catcatcatg gtttgtagaa agggtgagtg ggttgctttg 240aacccattga gaaagtgtca gaaaagacca tgtggtcacc caggtgatac tccattcggt 300actttcactt tgactggtgg taacgttttc gagtacggtg ttaaggctgt ttacacttgt 360aacgagggtt accagttgtt gggtgagatc aactacagag agtgtgatac tgacggttgg 420actaacgaca ttccaatctg tgaggttgtt aagtgtttgc cagttactgc tccagagaac 480ggtaagattg tttcctccgc tatggaacca gatagagagt accacttcgg tcaggctgtt 540agattcgttt gtaactccgg ttacaagatt gaaggtgacg aagagatgca ctgttctgat 600gacggtttct ggtccaaaga aaagccaaag tgtgttgaga tttcctgtaa gtccccagac 660gttattaacg gttccccaat ctcccaaaag atcatctaca aagagaacga gagattccag 720tacaagtgta acatgggtta cgagtactct gaaagaggtg acgctgtttg tactgaatct 780ggttggagac cattgccatc ctgtgaagag aagtcctgtg acaacccata cattccaaac 840ggtgactact ccccattgag aatcaagcac agaactggtg acgagatcac ttaccagtgt 900agaaacggtt tctacccagc tactagaggt aacactgcta agtgtacttc cactggttgg 960attccagctc caagatgtac tttgaagcca tgtgactacc cagatatcaa gcacggtggt 1020ttgtaccacg agaacatgag aagaccatac ttcccagttg ctgttggaaa gtactactcc 1080tactactgtg acgaacactt cgaaactcca tctggttctt actgggacca catccactgt 1140actcaagatg gttggtcccc agctgttcca tgtttgagaa aatgttactt cccatacttg 1200gagaacggtt acaaccagaa ctacggtaga aagttcgttc agggaaagtc cattgacgtt 1260gcttgtcatc caggttacgc tttgccaaag gctcagacta ctgttacttg tatggaaaac 1320ggttggtccc ctactcctag atgtatcaga gttaagactt gttccaagtc ctccatcgac 1380attgagaacg gtttcatttc cgagtcccag tacacttacg ctttgaaaga gaaggctaag 1440taccagtgta aattgggata cgttactgct gacggtgaaa cttccggttc catcacttgt 1500ggtaaggatg gttggtctgc tcaaccaact tgtatcaagt cttgtgacat cccagttttc 1560atgaacgcta gaactaagaa cgacttcaca tggttcaagt tgaacgacac tttggactac 1620gaatgtcacg acggttacga atctaacact ggttccacta ctggttccat cgtttgtggt 1680tacaacggtt ggtctgactt gccaatctgt tacgagagag agtgcgagtt gccaaagatc 1740gacgttcatt tggttccaga cagaaagaag gaccagtaca aggttggtga ggttttgaag 1800ttctcctgta agccaggttt cactatcgtt ggtccaaact ccgttcagtg ttaccatttc 1860ggtttgtccc cagacttgcc tatttgtaaa gagcaggttc agtcttgcgg tccaccacca 1920gaattgttga acggtaacgt taaagaaaag actaaagaag agtacggtca ctctgaggtt 1980gttgagtact actgtaaccc aagattcttg atgaagggtc caaacaagat ccaatgtgtt 2040gacggtgagt ggactacttt gccagtttgt atcgttgaag agtccacttg tggtgacatt 2100ccagaattgg aacacggttg ggctcaattg tcatccccac catactacta cggtgactcc 2160gttgagttca actgttccga gtccttcact atgattggtc acagatccat cacatgtatc 2220cacggtgttt ggactcaatt gccacagtgt gttgctatcg acaagttgca acaatgtcaa 2280tcctccaact tgatcatctt ggaggaacac ttgaagaaca agcaagagtt cgaccacaac 2340tccaacatcc aataccaatg tcaaggtcaa gagggttgga ttcacactgt ttgtatcaac 2400ggtcaatggg accctgaagt taactgttcc atggctcaga ttcagttgtg tccaccacct 2460ccacaaattc caaactccca caacatgact actactttga actacagaga tggtgagaag 2520gtttccgttt tgtgtcaaga gaactacttg atccaagagg gtgaggaaat cacttgtaag 2580gacggtagat ggcaatccat cccattgtgt gttgagaaga tcccatgttc ccaaccacca 2640caaattgagc acggtactat caactcttcc agatcctctc aagagtctta cgctcacggt 2700actaagttgt cctacacttg tgagggtggt ttcagaatct ctgaggaaaa cgagactact 2760tgttacatgg gaaagtggtc ctctccacca caatgtgaag gtttgccttg taagtctcca 2820ccagagattt ctcacggtgt tgttgctcac atgtccgact cttaccaata cggtgaagag 2880gttacttaca agtgtttcga gggtttcggt attgatggtc cagctatcgc taagtgtttg 2940ggtgaaaagt ggtcccatcc tccatcctgt atcaagactg actgtttgtc cttgccatct 3000ttcgagaacg ctatcccaat gggtgaaaag aaggacgttt acaaggctgg tgaacaggtt 3060acatacactt gtgctactta ctacaagatg gacggtgctt ccaacgttac ttgtatcaac 3120tccagatgga ctggtagacc aacttgtaga gacacttcct gtgttaaccc accaactgtt 3180cagaacgctt acatcgtttc cagacagatg tctaagtacc catccggtga gagagttaga 3240taccaatgta gatccccata cgagatgttc ggtgacgaag aggttatgtg tttgaacggt 3300aattggactg aaccaccaca gtgtaaggac tccactggta agtgtggtcc acctccacca 3360attgacaacg gtgacatcac ttctttccca ttgtccgttt acgctccagc ttcttccgtt 3420gagtaccagt gtcagaactt gtaccagttg gagggtaaca agagaatcac ttgtagaaac 3480ggacaatggt ctgagccacc aaagtgtttg cacccatgtg ttatctccag agaaatcatg 3540gaaaactaca acattgcttt gagatggact gctaagcaga agttgtactc cagaacaggt 3600gagtctgttg agtttgtttg taagagaggt tacagattgt cctccagatc ccacactttg 3660agaactacat gttgggacgg aaagttggag tacccaactt gtgctaagag ataatgagcg 3720gccgcttaat taa 3733

Claims

1.-20. (canceled)

21. A method for making a recombinant mammalian factor H (FH), the method comprising the step of: expressing a codon-optimized nucleic acid sequence encoding the mammalian FH in cells in culture, wherein the codon-optimized nucleic acid sequence comprises a sequence at least 80% identical to any one of SEQ ID NO: 1-13 or at least 80% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains, and wherein the nucleic acid sequence has been codon optimized to enhance expression in the cells and inserted into an expression vector.

22. The method according to claim 21, wherein at least 1 mg of the mammalian FH or variants thereof is produced per liter of culture medium.

23. The method according to claim 21 further comprising the step of purifying the mammalian FH or variant thereof from the cell and/or culture medium in which the cells are grown.

24. The method according to claim 21, wherein the cell is a yeast cell, and wherein the yeast cell is Pichia pastoris.

25. The method according to claim 21, wherein the mammalian FH is selected from the group consisting of human FH, mouse FH, rat FH, hamster FH, rabbit FH, dog FH, horse FH, cow FH, pig FH, sheep FH, camel FH, cat FH, and guinea pig FH.

26. The method according to claim 21, wherein the codon-optimized nucleic acid sequence includes one or more single-nucleotide polymorphisms.

27. The method according to claim 27, wherein the one or more single-nucleotide polymorphisms are selected from the group consisting of Ile62Val, Tyr402H is, and Arg1210Cys.

28. The method according to claim 21, wherein the protein encoded by the codon-optimized nucleic acid sequence includes one or more covalently modified natural or non-naturally encoded variant amino acids.

29. The method according to claim 28, wherein said one or more covalently modified natural or non-naturally encoded variant amino acids is covalently modified to include a moiety selected from the group consisting of glycosaminoglycans, polysialic acids, dextran (-1, 6 polyglucose), dextran (-1, 4 polyglucose), hyaluronic acid, chitosans, linear or branched polyethylene glycols, polyether polyols, N-(2-hydroxypropyl) methacrylamide copolymers, poly(vinylpyrrolidone), poly(ethyleneimine), linear polyamidoamines, poly(L-lysine), poly(glutamic acid), poly(malic acid) and poly(aspartamides).

30. The method according to claim 21, wherein protein encoded by the codon-optimized nucleic acid sequence is conjugated with a chemical moiety or chemical moieties, wherein the moiety or moieties improve the biotherapeutic properties of the codon-optimized nucleic acid sequence.

31. The method according to claim 21, wherein the deletion variant of the encoded protein comprises a deletion of two or more domains that is between 2 domains and 14 domains.

32. The method according to claim 21, wherein the codon-optimized nucleic acid sequence comprises a sequence at least 90% identical to any one of SEQ ID NO: 1-13 or at least 90% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains.

33. The method according to claim 21, wherein the protein encoded by the codon-optimized nucleic acid sequence retains mammalian FH-like biological activity.

34. The method according to claim 33, wherein the mammalian FH-like biological activity is assessed according to a biological activity assay, wherein the biological activity assay is selected from the group consisting of: (i) ability of the protein encoded by the codon-optimized nucleic acid sequence to act as a cofactor for factor I-catalysed cleavage of C3b; (ii) ability of the protein encoded by the codon-optimized nucleic acid sequence to promote acceleration of decay of C3bBb assembled on a surface plasmon resonance (SPR) sensor chip; (iii) affinity of the protein encoded by the codon-optimized nucleic acid sequence for C3b immobilized on a sensor chip; (iv) affinity of the protein encoded by the codon-optimized nucleic acid sequence for glycosaminoglycans (GAGs); or (v) ability of the protein encoded by the codon-optimized nucleic acid sequence to protect sheep erythrocytes from complement-mediated hemolysis by FH-depleted human sera.

35. A nucleic acid sequence capable of expressing a FH polypeptide or variant thereof, wherein the nucleic acid sequence is codon optimized for expression in a cell in culture in an amount greater than 1 mg per liter of culture, and wherein the nucleic acid sequence comprises a sequence at least 80% identical to any one of SEQ ID NO: 1-13 or at least 80% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains.

36. The nucleic acid sequence of claim 35 wherein nucleic acid sequence comprises a sequence at least 90% identical to any one of SEQ ID NO: 1-13 or at least 90% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains.

37. A vector comprising the nucleic acid sequence according to claim 35.

38. A recombinant version of mammalian FH produced as a result of the expression of the nucleic acid sequence of claim 35.

39. A method of treating or preventing a disease in a subject in need thereof comprising the step of administering the recombinant version of mammalian FH according to claim 38 to the subject.

40. The method according to claim 39 for use in treating or preventing a disease selected from the group consisting of AMD, atypical haemolytic uraemic syndrome (aHUS), or dense deposit disease (DDD).

Images