RECOMBINANT EXPRESSION OF MULTIPROTEIN COMPLEXES USING POLYGENES

Abstract

The present invention relates to a recombinant polynucleotide encoding a polygene coding for at least three polypeptides wherein at least one of the genes constituting the polygene is of non-viral origin, at least two of the polypeptides encoded by the genes constituting the polygene are each capable of at least transiently interacting with at least one other polypeptide encoded by a gene of said polygene, and the genes constituting the polygene are each connected to one another by a sequence coding for at least one protease cleavage site. The present invention also relates to polyproteins encoded by the polygene. Further embodiments of the present invention are a vector containing the recombinant polypeptide, a host cell containing the recombinant polypeptide and/or the vector and a non-human transgenic animal transformed with the recombinant polypeptide and/or the vector. The present invention also relates to methods for the production of the polynucleotide and for the manufacture of multiprotein complexes. The embodiments of the present invention are particularly useful in gene therapy, drug candidate screening, vaccine production and crystallisation of multiprotein complexes for structural investigations.

Description

[0001] The present invention relates to a recombinant polynucleotide encoding, each within a single open reading frame (ORF), at least two polygenes each coding for at least three biologically active polypeptides wherein at least two of the polypeptides encoded by the genes constituting the polygenes are of non-viral origin, at least two of the polypeptides encoded by the genes constituting the polygenes are each capable of at least transiently interacting with at least one other polypeptide encoded by a gene of said polygenes, and the genes constituting each polygene are connected to one another by a sequence coding for at least one protease cleavage site and/or by a sequence coding for at least one self-cleaving peptide. Further embodiments of the present invention are a vector containing the recombinant polynucleotide, a host cell containing the recombinant polynucleotide and/or the vector and a non-human transgenic animal transformed with the recombinant polynucleotide and/or the vector. The present invention also relates to methods for the production of the polynucleotide and for the manufacture of multiprotein complexes. The embodiments of the present invention are particularly useful in gene therapy, drug candidate screening, vaccine production and crystallisation of multiprotein complexes for structural investigation.

[0002] An intense focus of biological research efforts in the post-genomic era is the elucidation of protein interaction networks (interactome). Since many of the identified multiprotein complexes are not present in sufficient quantities in their native cells for detailed molecular biological analysis, their study is dependent on recombinant technologies for large-scale heterologous protein production. Recombinant expression methods require a disproportionate investment in both labor and materials prior to multiprotein expression, and subsequent to expression do not provide flexibility for rapidly altering the multiprotein components for revised expression studies.

[0003] There are several recombinant technologies that are currently used to obtain multisubunit complexes. Proteins can for example be expressed in isolation in E. coli either in soluble form or as inclusion bodies, purified and then reconstituted with similarly produced proteins in vitro into multiprotein complexes. Eukaryotic cells (e.g. mammalian or yeast cells) can also be used as hosts in transient expression experiments. This methodology is entirely dependent on the existence of an efficient in vitro reconstitution protocol. While this strategy may yield acceptable results for more simple systems with small subunit sizes, it is generally not applicable for more complicated multiprotein complexes containing many, and also large, subunits (e.g. close to all higher eukaryotic--in particular human--regulatory complexes).

[0004] Co-expression has been recognised as a superior alternative to the strategy of in vitro reconstitution as outlined above. Several co-expression systems have been developed in the past both for prokaryotic and eukaryotic expression. In prokaryotic systems, co-expression can be achieved by generating a single plasmid containing all genes of choice or by co-transforming several plasmids containing one or two genes and different resistance markers and replicons.

[0005] Co-expression in eukaryotic cells has been realised by using the baculovirus system, initially with limited success by co-infection with several viruses, and later and more successfully by expressing all proteins from a single virus, offering many advantages and eliminating several limitations present in prokaryotic systems (such as comparatively small subunit sizes, lack of authentic processing, difficult expression of eukaryotic (especially human) proteins etc.). For the baculovirus system, expression from a single virus has been shown to increase yields dramatically (Berger et al. (2004) Nature Biotech. 22, 1583-1587; see also Comment (2004), Nature Biotech. 22, vii, New & Views (2004), Nature Biotech. 22, 152, Research Highlights, Nature Methods 2, 7 (2005); Bertolotti-Ciarlet et al. (2003) Vaccine 21, 3885-3900), while decisively reducing the logistic demands especially for large scale production.

[0006] A major improvement of multiprotein expression was the provision of the modular system for the generation of multigene expression cassettes provided by the present inventors, which is disclosed in WO 2005/085456 A1 (PCT/EP2004/013381; see also Berger et al. (2004) ibid.). The MultiBac technology described in WO 2005/085456 A1 (PCT/EP2004/013381) enables the simple generation of multigene expression cassettes as well as modification and revision of expression experiments (Berger et al. (2004) ibid.).

[0007] However, a hindrance for successful expression and in vivo assembly of multisubunit complexes, in particular with many (6, 7, 8 or more), subunits (which constitute the majority of eukaryotic, e.g. human, gene regulatory complexes) is found in the fact that the relative expression levels of these subunits typically vary significantly based on in many cases not fully understood mechanisms (e.g. transcription and translation efficacy, protein stability, mRNA stability and secondary structures etc.). As a consequence, the subunit which is expressed in the least amount in an intrinsically unbalanced system will dictate the overall success of the multisubunit complex production experiment by limiting total complex yield. Accordingly, the transcription/translation machinery will produce excess amounts of other components which are not incorporated in the process thus "wasting" cellular transcription/translation resources. Individual expression levels typically vary several fold (e.g. up to 10 fold or more) with respect to each other, entailing losses which are refractory to a successful production of the desired multisubunit complexes, in particular in the case of complexes with more than 4, such as 5, 6, 8, 10 or more subunits (e.g. in the case of many eukaryotic gene regulatory complexes).

[0008] Viruses of the picornavirus super-group have a genome consisting of a single-stranded RNA molecule in sense orientation containing a single or two ORFs that code(s) for a polyprotein comprising the viral proteins which are connected to one another by cleavage sites of a viral protease or by self-cleaving peptides (reviewed, e.g., in Ryan et al. (1997) J. Gener. Virol. 78, 699-723).

[0009] The general concept of expressing a polyprotein through a recombinant virus for the production of protein complexes has been applied in the reconstitution of a TCR (T cell receptor):CD3 complex (Szymczak et al. (2004) Nature Biotech. 5, 589-594). The authors used two recombinant retroviral vectors wherein one vector contained the sequences encoding the two TCR subunits whereas the other vector encoded a polyprotein comprising the four CD3 subunits. The subunits were connected by self-cleaving 2A peptide sequences derived from aphthoviruses. One disadvantage of this approach is that, in order to reconstitute the complete complex, two separate vectors must be prepared and two transfections are necessary.

[0010] The viral polyprotein approach has been applied in baculovirus expression systems for small constructs such as heterodimeric IL-12 (Kokuho et al. (1999) Vet. Immunol. Immunopathol. 72, 289-302) and fusion proteins comprising a nuclear targeting signal derived from baculoviral polyhedrin and a protein of interest (U.S. Pat. No. 5,179,007).

[0011] Therefore, the technical problem underlying the present invention is to provide a new system for improved expression of multiple proteins.

[0012] The solution of the above technical problem is provided by the embodiments defined in the claims.

[0013] In particular, the present invention provides a polynucleotide encoding, each within a single open reading frame (ORF), at least two polygenes each coding for at least three biologically active polypeptides wherein at least two of the polypeptides encoded by the genes constituting the polygenes are of non-viral origin, at least two of the polypeptides encoded by the genes constituting the polygenes are each capable of at least transiently interacting with at least one other polypeptide encoded by a gene of said polygenes, and the genes constituting each polygene are connected to one another by a sequence coding for at least one protease cleavage site and/or by a sequence coding for at least one self-cleaving peptide.

[0014] The polynucleotide according to the present invention may be a DNA, RNA or a polynucleotide comprising one or more synthetic nucleotide analogues. The polynucleotide may be present in single or double stranded form. DNA, in particular double-stranded DNA, forms are especially preferred. The polynucleotide of the present invention may be produced by chemical synthesis. Preferred polynucleotide constructs of the present invention are made by recombinant gene technology (see, e.g., Sambrook et al. "Molecular Cloning", Cold Spring Harbor Laboratory, 1989).

[0015] A "polygene" as used herein is a nucleic acid sequence that encodes at least three biologically active polypeptides in a single ORF. Thus, each "gene" constituting the polygene is a nucleic acid sequence coding for a polypeptide, in particular a protein or fragment, variant, mutant or analogue thereof, having a specific, in particular structural, regulatory or enzymatic, function. Preferably the "gene" encoding the polypeptide comprises the coding region of a cDNA encoding the structural, regulatory or enzymatic protein or fragment, variant, mutant or analogue thereof.

[0016] A "fragment" of the polypeptide encoded by a gene contained in the polygenes means a part or region of the original polypeptide, preferably a fragment retaining at least one of the functions of the complete protein. A "variant" of the polypeptide encoded by a gene contained in the polygene means a polypeptide that is a functional or non-functional equivalent of the original polypeptide derived from another species or a functional or non-functional derivative of the original polypeptide that arises from alternative splicing or post-translational processing. A "mutant" of the polypeptide encoded by a gene contained in the polygene means a polypeptide that is derived from a naturally occurring protein by insertion, substitution, addition and/or deletion of one or more amino acid residues. An "analogue" of the polypeptide encoded by a gene contained in the polygene means functional equivalent of the original polypeptide that may even have a non-related amino acid sequence but exerts the same function as the polypeptide it is analogous to.

[0017] Correspondingly, on the nucleic acid level, a gene "fragment" is a part or region of the original gene the "fragment" is derived from. The gene "variant" has a sequence that is found in a different species compared to the original gene, or it may encode a splicing variant or post-translationally processed version of the polypeptide in question. The "mutant" is derived from the parent gene by insertion, substitution, addition and/or deletion of one or more nucleotides. The "analogue" of a gene encodes a functional equivalent of the polypeptide encoded by the parent gene.

[0018] At least two of the genes in the polygenes according to the present invention are of non-viral origin. "Non-viral" means that the nucleic acid sequence encoding the polypeptide (representing a functional protein or a fragment, variant, analogue or mutant thereof) is originally not found in or not derived from the genome of a virus. In particular, nucleotide sequences comprised in the polygene of the polynucleotide according to the present invention stem from eukaryotes and/or prokaryotes.

[0019] Thus, according to the present invention, the genes encoding the subunits (such as a multiprotein complex or members of a metabolic pathway or any other proteins that at least potentially interact at least transiently with one another) of a multisubunit assembly are present in at least two open reading frames (ORFs). The sequences encoding the subunits (polypeptides) of the assembly are present in at least two polygenes wherein the genes constituting each polygene are connected to one another by a sequence (there may be more than one) coding for a protease cleavage site (i.e. a sequence comprising the recognition site of a protease) or at least one self-cleaving peptide.

[0020] According to a preferred embodiment of the present invention the protease(s) capable of cleaving the cleavage sites encoded by the sequence(s) connecting the genes constituting the polygenes is/are encoded by the polynucleotide of the present invention. More preferably, the gene(s) encoding the protease(s) is/are part of at least one of the polygenes.

[0021] Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan et al. (1997) J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Preferred examples of protease cleavage sites are the cleavage sites of potyvirus Nla proteases (e.g. tobacco etch virus protease), potyvirus HC proteases, potyvirus P1 (P35) proteases, byovirus Nla proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3C proteases, connovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, thrombin, factor Xa and enterokinase. Due to its high cleavage stringency, TEV (tobacco etch virus) protease cleavage sites are particularly preferred. Thus, the genes of the polygenes according to the present invention are preferably connected by a stretch of nucleotides comprising a nucleotide sequence encoding an amino acid sequence of the general form EXXYXQ(G/S) wherein X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S). Most preferred are linker nucleotide sequences coding for ENLYFQG and ENLYFQS, respectively.

[0022] Preferred self-cleaving peptides (also called "cis-acting hydrolytic elements", CHYSEL; see deFelipe (2002) Curr. Gene Ther. 2, 355-378) are derived from potyvirus and cardiovirus 2A peptides. Especially preferred self-cleaving peptides are selected from 2A peptides derived from FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus and porcine teschovirus.

[0023] At least two of the polypeptides encoded by the polygenes of the present invention are capable of at least transiently interacting with one other polypeptide encoded by the polygenes, or they are at least suspected to be capable of at least transiently interacting with another polypeptide encoded by a gene contained in the polygenes. Typical "interactions" formed between the polypeptides include covalent binding, hydrogen bonds, electrostatic interactions and Van-der-Waals interactions. "Transient" interactions are common to biomolecules, in particular proteins, and are typically represented by interactions between enzymes and their substrates, receptors and their (agonistic or antagonistic) ligands, interactions between members of metabolic pathways and interactions between proteins of regulatory (e.g. gene regulatory) complexes.

[0024] The polypeptides encoded by the nucleotide sequences constituting the polygenes of the present invention may be the same or different. Thus, each polygene present in the constructs of the invention may contain one or more copy of each nucleotide sequence encoding a protein of interest. In this manner it is, e.g., possible to provide constructs that serve for optimal expression of the desired proteins, in particular in case proteins are normally expressed at different levels and/or are present in a macromolecular assembly in different stoichiometries. Therefore, in case a polypeptide is poorly expressed in commonly used systems, two or more copies of the corresponding coding sequence may be integrated into one or more polygene(s) of an inventive construct. The same approach may be used, in case a polypeptide is present as a dimer, trimer or multimer in a desired complex. In this manner, the constructs of the present invention may be assembled individually according to the requirements (expression levels, stoichiometry etc.) of any complex or other macromolecular assembly a person skilled in the art desires to express and/or to purify.

[0025] It is further preferred that the genes constituting the polygenes are selected from the group consisting of genes encoding members of multiprotein complexes and genes encoding members of metabolic pathways. Preferred multiprotein complexes are gene regulatory protein complexes such as transcription factor complexes, transport complexes such as complexes involved in nuclear and/or cellular transport, protein folding complexes, receptor/ligand complexes, cell-cell recognition complexes, complexes involved in apoptosis, complexes involved in cell cycle regulation etc. Members of metabolic pathways are, e.g. members of carbohydrate metabolism (such as glycolysis, gluconeogenesis, citric acid cycle, glycogen biosynthesis, galactose pathway, calvin cycle etc.), lipid metabolism (such as triacylglycerol metabolism, activation of fatty acids, .beta.-oxidation of fatty acids (even chain/odd chain), .alpha.-oxidation pathway, fatty acid biosynthesis, cholesterol biosynthesis etc.), amino acid metabolism such as glutamate reactions, Krebs-Henseleit urea cycle, shikimate pathway, Phe and Tyr biosynthesis, Trp biosynthesis etc.), energy metabolism (such as oxidative phosphorylation, ATP synthesis, photosynthesis, methane metabolism etc.) nucleic acid metabolism (purin and pyrimidine biosynthesis and degradation, DNA replication etc.). Members of multiprotein complexes and members of metabolic pathway may be taken from, e.g. http://www.biocarta.com/genes/index.asp and G. Michal (ed.) Biochemical Pathways, 1. edition, John Wiley & Sons, Hoboken, N.J., USA, 1999, the disclosure content of which is hereby incorporated by reference.

[0026] Each polygene according to the present invention contains at least 3 genes, i.e. sequences encoding a biologically active polypeptide. More preferred are polygenes encoding 4, 5 6 or more or even more proteins. As mentioned above, it is preferred that the protease(s) capable of cleaving the protease cleavage sites connecting the polypeptides is/are encoded by at least one of the polygenes.

[0027] According to a preferred embodiment, the polynucleotide of the present invention contains at least two promoter sequences which are each operatively linked to one of the polygenes, thus capable of controlling the expression of the polygenes. Suitable promoters in the constructs of the present invention may be selected from the group consisting of polh, p10 and pXIV very late baculoviral promoters, vp39 baculoviral late promoter, vp39polh baculoviral late/very late promoter, P.sub.cap/polh, pcna, etl, p35, da26 baculoviral early promoters; CMV, SV40, UbC, EF-1.alpha., RSVLTR, MT, P.sub.DS47, Ac5, P.sub.GAL and P.sub.ADH. The promoter sequences may be the same for all polygenes, or different promoters may be selected for the different polygenes.

[0028] Preferably, the each ORF containing a polygene of the present invention is flanked by a terminator sequence such as SV40, HSVtk or BGH (bovine growth hormone).

[0029] The polynucleotide according the present invention may contain further regulatory sequences such as enhancers or suppressor sequences.

[0030] It is further preferred that the polynucleotide according to the present invention contains at least one site for its integration into a vector or host cell. Such an integration site will allow for the convenient genomic or transient incorporation of the polynucleotide into vectors (such as virus) and host cells (e.g. eukaryotic host cells), respectively. Sites for genomic integration are more preferred.

[0031] Especially preferred integration sites are those which are compatible for the polynucleotide's integration into a virus. More preferably, the integration site is compatible for the polynucleotide's integration into a virus selected from the group consisting of adenovirus, adeno-associated virus (AAV), autonomous parvovirus, herpes simple virus (HSV), retrovirus, rhadinovirus, Epstein-Barr virus, lentivirus, semliki forest virus and baculovirus.

[0032] In a further preferred embodiment, the integration site is compatible for the polynucleotide's integration into a eukaryotic host cell which may preferably be selected from the group consisting of mammalian (such as human cells, e.g. HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32, MT-2, pancreatic .beta. cells, keratinocytes, bone-marrow fibroblasts, CHP212, primary neural cells, W12, SK-N-MC, Saos-2, WI38, primary hepatocytes, FLC3, 143TK--, DLD-1, umbilical vein cells, embryonic lung fibroblasts, primary foreskin fibroblasts, osteosarcoma cells, MRC5, MG63 cells etc.), porcine (such as CPL, FS-13, PK-15) cells, bovine (such as MDB, BT) cells, ovine (such as FLL-YFT) cells, C. elegans cells, yeast (such as S. cerevisiae, S. pombe, C. albicans, P. pastoris) cells, and insect cells (such as S. frugiperda, e.g. Sf9, Sf21, Express Sf+, High Five H5 cells, D. melanogaster, e.g. S2 Schneider cells).

[0033] Particularly preferred integration sites are selected from the transposon elements of Tn7, .lamda. integrase-specific attachment sites and SSRs (site specific recombinases), preferably the cre-lox specific (LoxP) site or the FLP recombinase specific recombination (FRT) site.

[0034] In a preferred embodiment of the present invention the polynucleotide additionally comprises one or more resistance markers for selecting host cells with desired properties based on a resistance to otherwise toxic substances. Examples of suitable resistance markers are those providing resistance against ampicillin, chloramphenicol, gentamycin, spectinomycin and/or kanamycin.

[0035] For its incorporation into a prokaryotic host cell, the polynucleotide of the present invention preferably comprises a conditional R6K.gamma. origin of replication for making propagation dependent on the pir gene in a prokaryotic host.

[0036] Especially preferred embodiments of the polynucleotide of the present invention result by the insertion of the polygenes as expression cassettes into constructs disclosed in WO 2005/085456 A1 (PCT/EP2004/013381).

[0037] Therefore, it is preferred that the polynucleotide of the present invention comprises a functional arrangement according to the following Formula I

X-T1-MCS1-P1-[A-B]-P2-MCS2-T2-Y (I)

comprising

[0038] (a) at least two expression cassettes T1-MCS1-P1 and P2-MCS1-T2 in a head-to-head, head-to-tail or tail-to-tail arrangement, each comprising a multiple cloning site MCS1 or MCS2, flanked by a promoter P1 and a terminator sequence T1 for MCS1 and flanked by a promoter P2 and a terminator sequence T2 for MCS2

[0039] (b) at least one multiplication module M in between the promoters P1 and P2 comprising at least two restriction sites A and B

[0040] (c) at least two restriction sites X and Y each flanking one of the expression cassettes, wherein

[0041] (i) restriction sites A and X as well as B and Y are compatible, but

[0042] (ii) the ligation products of AY and BX are not enzymatically cleavable by restriction enzymes a, b, x or y specific for restriction sites A, B, X and Y, and

[0043] (iii) restriction sites A and B as well as restriction sites X and Y are incompatible, wherein each polygene is inserted into one of the expression cassettes.

[0044] With respect to further preferred embodiments of the constructs having the arrangement of Formula (I) it is expressly referred to WO 2005/085456 A1 (PCT/EP2004/013381). In particular, restriction sites A and B in the multiplication module M are selected from the group consisting of restriction sites BstZ171, Spel, Clal and Nrul or restriction sites cleaved by isoschizomers thereof. Isoschizomers are restriction enzymes that have identical cleavage sites. Furthermore, preferred examples of the restriction sites X and Y are restriction sites selected from the group consisting of Pmel and AvrII or restriction sites cleaved by isoschizomers thereof.

[0045] Particularly preferred polynucleotides having each polygene inserted into one of the expression cassettes contained in the above formula (I) comprise the following features:

[0046] (a) promoters P1 and P2 are selected from the group consisting of polh and p10;

[0047] (b) terminator sequences are selected from the group consisting of SV40 and HSVtk;

[0048] (c) restriction sites A and B in the multiplication module M are selected from group consisting of restriction sites BstZ171l, Spel, Clal and Nrul;

[0049] (d) restriction sites X and Y are selected from the group consisting of restriction sites Pmel and AvrII; and

[0050] (e) sites for virus integration are selected from the group consisting of cre-lox and Tn7.

[0051] With respect to the production of polynucleotides having the above arrangement according to Formula (I) it is expressly referred to WO 2005/085456 A1 (PCT/EP2004/013381).

[0052] The provision of polynucleotides of the present invention encoding several biologically active polypeptides within two or more ORFs each containing a polygene provides a major improvement with respect to the cloning and expression of genes coding for members of multisubunit protein complexes: On the one hand, assembly of all subunit genes into a single ORF is often impossible or highly difficult because of the huge size and numbers of coding sequences to be coupled. On the other hand, efficient assembly of several or all members of a multisubunit complex each present in separate expression cassettes has often turned out to be highly inefficient, since the overall complex yield is determined by the least expressed subunit. According to the present invention the subunits of a multiprotein assembly are encoded by at least two polygenes (each representing a single ORF) each coding for at least three polypeptides (preferably of non-viral origin) which results in an optimal compromise between manageability of the construct and its constituents (in particular assembly of the polygenes) and expression efficiency (in particular in case the polynucleotide of the present invention is present in a suitable vector).

[0053] Therefore, a further embodiment of the present invention is a vector containing the above-described polynucleotide. The vector may be selected from the group consisting of plasmids, expression vectors and transfer vectors. More preferably, the vector of the present invention is useful for eukaryotic gene transfer, transient or viral vector-mediated gene transfer.

[0054] Especially preferred vectors are eukaryotic expression vectors such as viruses selected from adenovirus, adeno-associated virus (AAV), autonomous parvovirus, herpes simple virus (HSV), retrovirus, rhadinovirus, Epstein-Barr virus, lentivirus, semliki forest virus and baculovirus. Most preferred vectors of the present invention are baculovirus expression vectors. Preferred baculovirus of the present invention are embodiments wherein the genes v-cath and chiA are functionally disrupted, since this leads to improved maintenance of cellular compartments during infection and protein expression. The v-cath gene encodes the viral protease V-CATH which is activated by upon cell death by a process dependent on a juxtaposed gene on the viral DNA, chiA, which codes for a chitinase. Both genes are preferably disrupted to eliminate V-CATH activity and to gain the option of utilising chitin affinity chromatography without interference form the chiA gene product. The quality of the expression products generated by a baculovirus system lacking functionally active v-cath and chiA genes is significantly improved because of the reduction of viral-dependent proteolytic activity and cell lysis.

[0055] Preferably, vectors according to the present invention comprise a site for SSRs, preferably LoxP for cre-lox site specific recombination. More preferably, the cre-lox site is located in one or both of the baculoviral gene v-cath and chiA so as to disrupt their function.

[0056] The vector of the present invention preferably contains one ore more marker genes for selection of hosts successfully transfected with the correctly assembled vector. Examples of suitable marker genes are luciferase, .beta.-Gal, CAT, genes encoding fluorescent proteins such as GFP, BFP, YFP, CFP and variants thereof, and the lacZ.alpha. gene. The marker gene(s) may be functionally equivalent variants, mutants, fragments or analogues of the mentioned examples or other suitable markers known to the skilled person. Variants, mutants or analogues preferably show a homology of at least 75%, more preferably 85%, especially preferred 90%, in particular at least 95% on the amino acid level in comparison to the marker said variant, mutant or analogue is derived from.

[0057] In another preferred embodiment the vector of the present invention comprises a transposon element, preferably the Tn7 attachment site. More preferably, such a transposon element, e.g. the Tn7 attachment site, is located within a marker gene such that a successful integration by transposition can be assessed by testing the phenotype provided by the functional marker gene.

[0058] Preferred transfer vectors of the present invention are based on pFBDM or pUCDM as disclosed in WO 2005/085456 A1 (PCT/EP2004/13381; see SEQ ID NO: 1 and 2 as well as FIGS. 1 and 2, respectively disclosed therein). Further preferred transfer vectors of the present invention are based on derivatives of the above pFBDM and pUCDM, respectively, vectors.

[0059] Examples of particularly preferred derivatives of pFBDM and pUCDM are transfer vectors pSPL (FIG. 3), pFL (FIG. 4), pKL (FIG. 5) and pKDM (FIG. 6). Like pUCDM, pSPL contains a conditional origin of replication (R6K.gamma.). pFL (like pFBDM) contains a high copy-number replication origin (ColE1). pKDM and pKL have low-copy replication origins derived from pBR322. In analogy to pFBDM, pFL, pKL and pKDM contain transposon elements (Tn7R, Tn7L). Vectors pSPL, pFL and pKL have a LoxP imperfect inverted repeat flanking the dual expression cassette (as does pUCDM). All vectors contain the above-described multiplication module (M) for generating multigene cassettes1. pFL and pKL (and derivatives) are acceptor vectors, pUCDM and pSPL (and derivatives) are donor vectors in Cre-mediated plasmid fusions.

[0060] Important features of the above preferred examples of transfer vectors for generating the constructs of the present invention are summarised in the following Table 1.

TABLE-US-00001 TAB. 1 Features of preferred transfer vectors Recombination Antibiotic and resistance Replicon multiplication Vector marker (source) Host strain elements Usage pFBDM Ampicillin ColE1 TOP10* Tn7L, Tn7R, integration in Gentamycin multiplication MultiBac ** module M Tn7 site pFL Ampicillin ColE1 TOP10 Tn7L, Tn7R, acceptor for Gentamycin LoxP, plasmid multiplication fusions; module M integration in MultiBac ** Tn7 site pKDM Kanamycin pBR322 TOP10 Tn7L, Tn7R, integration in Gentamycin multiplication MultiBac ** module M Tn7 site pKL Kanamycin pBR322 TOP10 Tn7L, Tn7R, acceptor for Gentamycin LoxP, plasmid multiplication fusions; module M integration in MultiBac ** Tn7 site pUCDM Chloramphenicol R6K.gamma. BW23473 LoxP, donor for multiplication plasmid module M fusions; integration in MultiBac ** LoxP site pSPL Spectinomycin R6K.gamma. BW23473 LoxP, donor for multiplication plasmid module M fusions; integration in MultiBac ** LoxP site * or any other general laboratory cloning strain (recA.sup.- endA.sup.- pir.sup.-) ** see WO 2005/085456 A1

[0061] Therefore, the polygenes of the present invention are inserted into a vector such as pFBDM, pUCDM, pSPL, pFL, pKL or pKDM at the multiple cloning sites (MCS1 and MCS2), either by restriction enzyme cleavage and ligation or via recombination (e.g. using the BD In-Fusion enzyme). The baculovirus transfer vectors pFBDM, pUCDM, pSPL, pFL, pKL and pKDM comprise modified recipient baculovirus DNA engineered for improved protein production and allow for a simple and rapid method to integrate genes via two access sites (attTn7 and LoxP) into this baculoviral DNA in E. coli cells tailored for this purpose.

[0062] According to a further embodiment the present invention provides a host cell containing the polynucleotide and/or the vector of the invention.

[0063] Examples of preferred host cells are mammalian cells, such as human, rodent, porcine cells such as CPL, FS-13 and PK-15, bovine cells such as MDB and BT, ovine cells such as FLL-YFT, C. elegans cells, yeast cells such as S. cerevisiae, S. pombe, P. pastoris and C. albicans, insect cells such as cells from S. frugiperda, preferably Sf9, Sf21, Express Sf+ or High Five h5 cells, cells from D. melanogaster such as S2 Schneider cells, and bacteria such as E. coli, preferably strains Top10, Dh5.alpha., DH10.alpha., HB101, TG1, BW23473 and BW23474.

[0064] Preferred human cells are selected from HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32, MT-2, pancreatic .beta. cells, keratinocytes, bone-marrow fibroblasts, CHP212, primary neural cells, W12, SK-N-MC, Saos-2, WI38, primary hepatocytes, FLC3, 143TK--, DLD-1, umbilical vein cells, embryonic lung fibroblasts, primary foreskin fibroblasts, osteosarcoma cells, MRC5 and MG63 cells.

[0065] Host cells comprising a polynucleotide and/or vector according to the invention may be isolated cells or they may be present in tissues or organs.

[0066] A further embodiment of the present invention relates to a non-human transgenic animal being transformed with at least one polynucleotide sequence and/or vector of the invention. Preferred transgenic animals are rodent, porcine, bovine and C. elegans species.

[0067] The transgenic animal of the present invention is particularly useful for the elucidation of the role of multiprotein complexes or for screening of compounds for their biological activities in vivo.

[0068] A further embodiment of the present invention is a method for the production of the polynucleotide as defined above comprising the steps of:

[0069] (a) providing, preferably amplifying, the coding regions of the genes constituting the at least two polygenes;

[0070] (b) providing said coding regions with the sequences coding for the at least one protease cleavage site and/or the at least one self-cleaving peptide; and

[0071] (c) assembling the fragments resulting from steps (a) and (b) such that a single ORF results in each polygene; and

[0072] (d) combining the at least two polygenes into a single polynucleotide.

[0073] Another aspect of the invention is a method for the production of the vector according to the present invention comprising the steps of

[0074] (a) generating at least two polygenes each comprising at least three genes within a single ORF as defined above (preferably by the above method for the production of the polynucleotide of the present invention); and

[0075] (b) cloning the polygenes into a plasmid or viral vector, wherein at least one of the genes of each polygene is of non-viral origin and at least two of the polypeptide encoded by the genes are capable of at least transiently interacting with one other polypeptide encoded by the genes.

[0076] Preferably, one of the genes assembled into the polygenes is a gene encoding a protease capable of cleaving the protease cleavage sites connecting the polypeptides encoded by the polygenes. Preferred proteases are as defined above.

[0077] The construction of the polygenes as well as the vectors of the present invention can be carried out through various molecular biological techniques which are generally known to a person skilled in the art (see, e.g., Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, Hoboken, N.J., USA, 2003). The production of a polygene may be carried out, e.g. by PCR amplification of the nucleotide sequences coding for the particular polypeptides (e.g. using corresponding cDNA templates), preferably by usage of a primer (either 5' or 3') providing the sequence(s) coding for the at least one protease cleavage site and/or the at least one self-cleaving peptide. Preferably, the primers further contain a recognition sequence of a suitable restriction enzyme. Preferably, each primer contains a restriction site that is different from the restriction site of the other primer such that a directional ligation of the resulting amplification product with another amplification product and/or a linearised vector containing the same restriction sites is possible. According to another preferred embodiment for the production of constructs by directional ligation, the primers may contain a recognition sequence of a restriction enzyme that produces an overhang which is not self-ligatable such as Rsrll or BstEll. In case the primers themselves do not contain a restriction site, it is also possible to provide the amplification products with adapters comprising the desired restriction site(s). Of course, it is also possible to provide the coding regions for the desired polypeptides using any source (besides amplification). For example, the required sequences may be already present as such or in corresponding vectors from which the sequences may be cut out by appropriate restriction enzymes. Constructs that do not contain appropriate restriction sites etc. may be provided with the appropriate sequences (restriction site(s), protease cleavage site(s)/self-cleavable peptide(s), linkers etc.) by ligation of suitable adapters containing the required elements. The amplification products or any other appropriate construct (containing the restriction sites) are then cut with the appropriate restriction enzymes. Of course, the sequences of any primers used are preferably selected such that, after final assembly of the polygenes, preferably into a suitable vector, a single ORF results for each polygene.

[0078] According to a preferred embodiment of the invention, the amplification products or other appropriate sequences may then be ligated sequentially or simultaneously into a suitable vector, e.g. at MCS1 or MCS2 of the MultiBac vector system referred to above. For example, one polygene may be introduced into pFDBM and another polygene may be ligated into pUCDM (as described in WO 2005/085456 A1 (PCT/EP2004/013381)). The resulting constructs are then used for the production of corresponding bacmids by cre-lox site-specific recombination (pUCDM derivative) and Tn7 specific transposition (pFBDM derivative) yielding a baculoviral expression vector ready for infection of corresponding insect cells for expression and purification of the multiprotein complex of interest.

[0079] Besides producing the polygenes and introducing these into a suitable vector by restriction/ligation it is also possible to assemble such constructs by homologous recombination using appropriate recombinases. Suitable examples of recombinase-based cloning techniques are the In-Fusion.RTM. system available from BD Biosciences Clontech, Heidelberg, Germany (see Clontechniques, October 2002, p. 10) and the Red.RTM./ET.RTM. recombination system available from Gene Bridges GmbH, Dresden, Germany (see WO-A-99/29837; http://www.genebridges.com).

[0080] The In-Fusion.RTM. system requires 15 bp homologous regions in a DNA molecule to be fused into a linear construct (such as an appropriate vector) having the corresponding homologous regions. Accordingly, a vector containing a polygene of the present invention may assembled sequentially or simultaneously by providing the constituting coding regions (together with the linker sequences coding for a protease cleavage site and/or a self-cleaving peptide) with appropriate homologous sequences of 15 bp. If desired, the 15 bp homologous sequences may be selected such that the constituting coding regions are assembled in a desired order. Of course, appropriate homology regions may be introduced by PCR amplification of the corresponding fragments using primers containing the desired sequences.

[0081] The Red.RTM./ET.RTM. recombination system is different from the In-Fusion.RTM. system in that homology sequences of 40 to 60 base pairs are required, but the construct in which a fragment is to be inserted needs not to be linear. The recombination is carried out in vivo in a host, preferably an E. coli strain, expressing the dual recombinase system "ET" (RecE/RecT or Red.alpha./Red.beta.). Thus, the fragments constituting the polygenes may be directly transformed together with an appropriate vector into the appropriate host, preferably E. coli cells. In this manner, each polygene may be assembled in the suitable vector either sequentially fragment by fragment (preferably comprising the coding region for each member polypeptide of the multiprotein complex+ at least one protease cleavage site sequence/self-cleaving peptide sequence), or by simultaneous transformation of all fragments.

[0082] Especially preferred polynucleotide constructs of the present invention contain polygenes of at least similar length, since the inventors have found that the expression of corresponding polyproteins from such constructs results in comparable expression levels. According to the present invention "polygenes of similar length" means that the lengths of the nucleotide sequences of the polygenes differ from one another by not more than 50%, more preferably not more than 30%, in particular not more than 20% or even less.

[0083] Furthermore, the present invention provides a method for the production of multiprotein complexes in vitro comprising the steps of

[0084] (a) cultivating the host cell according to the present invention in a suitable medium under conditions allowing the expression of the polygenes; and

[0085] (b) recovering the expression products encoded by the polygenes from the medium and/or the host cells.

[0086] The present invention also relates to a method for the production of multiprotein complexes in vivo comprising the steps of

[0087] (a) generating at least two polygenes each comprising at least three genes within a single ORF as defined above; and

[0088] (b) transforming the polygenes into an animal such that the polygenes are expressed in said animal.

[0089] Preferably the transformation of the animal with the polygenes according to step (b) is effected by means of a vector, in particular a viral vector, more preferably a baculovirus vector. Baculoviruses are especially useful vehicles for delivery of polygenes into mammalian species. The above in vivo method is preferably carried out in mammals, C. elegans or insects. Particularly preferred examples of suitable animal species are defined herein above.

[0090] The embodiments of the present invention are also useful for the preparation of vaccines directed against multisubunit assemblies of proteins. Complexes of multiple subunits often display different epitopes compared to the individual proteins constituting the complexes. Therefore, the multiprotein complexes produced according to the present invention display the naturally occurring relevant epitopes in a more appropriate fashion, thus providing better antigen targets for antibody production.

[0091] Recently, virion-like particles (VLPs) consisting of four proteins from the sever acute respiratory syndrome (SARS) coronavirus were made using a recombinant baculovirus expression vector (cf. Mortola et al. (2004), FEBS Lett. 576, 174-178). The effective expression of such infectious particles for the preparation of vaccines will be greatly facilitated using the polygene expression system according to the present invention. In particular, the high-yield expression of multisubunit assemblies that contain substantially more polypeptide than the example of SARS-VLPs is made available by the expression tools of the present invention.

[0092] Therefore, the present invention further relates to a method for the production of a vaccine comprising the steps of

[0093] (a) administering at least one polynucleotide and/or vector of the present invention to a mammal, whereby the polygene of the invention is expressed within the mammal;

[0094] (b) optionally administering an adjuvans to the mammal; and

[0095] (c) optionally isolating the antibodies and/or spleen cells producing antibodies specific for at least one of the polypeptide encoded by the polygenes.

[0096] The present invention provides a convenient and simple approach for the recombinant production of multiprotein assemblies. These multisubunit assemblies may be tested for protein complex interactions or modifications of the proteins constituting the multisubunit assembly. The multisubunit assemblies produced according to the present invention may also be assayed for their interaction with candidate compounds (small organic molecules, nucleic acids, peptides, polypeptides etc.) that may exert a biologically significant activity being of medical value.

[0097] Therefore, the present invention is also directed to a method for assaying protein complex interactions or protein modifications.

[0098] According to a preferred embodiment, the present invention provides a method for the screening of protein complex interactions or modifications of multiprotein complexes in vitro comprising the steps of

[0099] (a) providing a host cell according to the present invention containing at least two polygenes;

[0100] (b) maintaining the host cell under conditions that allow expression of the polygenes; and

[0101] (c) detecting interactions between or modifications of the polypeptides encoded by the polygenes.

[0102] Another preferred embodiment of the present invention is a method for in vitro screening of candidate compounds capable of (i) interacting with a multiprotein complex and/or (ii) modification of proteins within a multiprotein complex and/or (iii) inhibiting interactions within or between multiprotein complexes and/or inhibiting modifications of proteins within a multiprotein complex, comprising the steps of

[0103] (a) providing a host cell according to the present invention containing at least two polygenes;

[0104] (b) maintaining the host cells under conditions that allow expression of the polygenes;

[0105] (c) contacting a candidate compound with the host cell; and

[0106] (d) detecting interactions of the expression products with the candidate compound and/or interactions between the expression products and/or modifications of the expression products and/or inhibition of interactions between the expression products.

[0107] The polynucleotides and/or vectors of the present invention are also suitable for the screening of protein-protein, protein-(multi)protein complex or multiprotein complex-multiprotein complex interactions or modifications (phosphorylation, glycosylation etc.) of multiprotein complexes in vivo.

[0108] Thus, a further preferred embodiment of the present invention is a method for in vivo screening of candidate compounds capable of (i) interacting with a multiprotein complex and/or (ii) modification of proteins within a multiprotein complex and/or (iii) inhibiting interactions within or between multiprotein complexes and/or inhibiting modifications of (a) proteins within a multiprotein complex, comprising the steps of

[0109] (a) providing an animal comprising at least one polynucleotide and/or vector of the invention containing at least two polygenes as defined above, whereby the polygenes are expressed in the animal;

[0110] (b) administering a candidate compound to the animal; and

[0111] (c) detecting interactions of the expression products with the candidate compound and/or interactions between the expression products and/or modifications of the expression products and/or inhibition of interactions between the expression products.

[0112] The multiprotein expression tools of the present invention are also of medical use. In particular, bioactive multiprotein complexes as well as medically advantageous combinations of proteins, e.g. antibody mixtures, optionally in combination with interleukins and/or adjuvans can be administered to an animal or human by means of the polynucleotides and/or the gene delivery vectors of the present invention.

[0113] Accordingly, the present invention further relates to the use of the polynucleotide and/or the vector and/or the host cell described above for the preparation of a medicament comprising a polygene transfer vehicle for gene therapy.

[0114] Tremendous efforts are being made to develop gene delivery systems for therapeutic applications. Gene therapy has been the focus of intense enthusiasm but also criticism in the past. To date, major progress has been made in evaluating gene therapy in clinical trials on the way to achieving safe and applicable clinical in vivo and ex vivo strategies for human diseases (see Worgall S. (2004) Peadiatr. Nephrol.). Overall, gene therapy now stands as a very promising avenue for the correction of genetic as well as acquired disorders entailing permanent or transient expression of a therapeutic gene product (Worgall S., ibid.). Recombinant vectors based on virus, in particular those that are not replication competent in mammalian hosts (e.g. baculoviral vectors) have emerged recently as a powerful tool for mammalian cell gene delivery and have been successfully applied to a whole range of mammalian cell lines including human, primate, rodent, bovine, procine and ovine cells (reviewed in Kost and Condreay (2002) Trends Biotech. 20, 173-180). To obtain complex gene transfer/therapy effects, both ex vivo and in vivo, an increasing demand has arisen for polycistronic viral vectors to accomplish more powerful results rather by combined gene therapy than by single gene therapy (de Felipe (2002), Curr. Gene Ther. 2, 355-378; Planelles (2003) Meth. Mol. Biol. 229, 273-284). The requirement for the incorporation of accessory genes into a carrier virus that is to be administered in vivo, e.g to block inactivation by the complement system, has also been demonstrated by using a pseudotyped baculovirus with baculoviral gp64 envelope proteins that carried a human decay-accelerating factor protein domain fusion (Hueser et al. (2001) Nat. Biotech. 19, 451-455), exemplifying the necessity to provide recombinant modifications on the virus production level in addition to the multiple genes to be transferred for therapeutic purposes.

[0115] Accordingly, recombinant baculovirus of the present invention are preferred for preparing gene therapeutic medicaments. More preferably, the vector used for the medicament of the present invention is a baculovirus comprising at least two polygenes as defined above encoding

[0116] (i) one or more therapeutic polypeptide(s) and

[0117] (ii) one or more baculoviral proteins

[0118] In a preferred embodiment, the protein(s) according to (ii) are humanised baculoviral proteins expressed from pseudotyped baculovirus, preferably a humanised baculovirus envelope protein gp64, e.g. gp64 fused with a human protein such as for example decay accelerating factor.

[0119] Furthermore, the present invention relates to an in vivo gene therapeutic method comprising the steps of

[0120] (a) providing a polygene transfer vehicle comprising a polynucleotide according to the invention; and

[0121] (b) administering the polygene transfer vehicle to a patient suffering from a genetic disorder.

[0122] The present invention further provides an ex vivo gene therapeutic method comprising the steps of

[0123] (a) collecting cells of a patient suffering from a genetic disorder;

[0124] (b) transforming the collected cells with a polygene transfer vehicle comprising a polynucleotide according to the present invention; and

[0125] (c) administering the transformed cells to the patient.

[0126] The multiprotein complexes produced according to the present invention may advantageously be used in biophysical studies, in particular structural studies using crystallographical, electron-microscopical and/or NMR techniques, protein chemical studies, in particular for protein-protein interactions, and for drug development.

[0127] Thus, the present invention is directed to the use of the polynucleotide and/or the vector and/or the host cell of the present invention for the crystallisation of multiprotein complexes.

[0128] A further embodiment of the present invention is a kit for the preparation of multiple-protein complexes comprising

[0129] (a) primers for PCR amplification of the coding sequences constituting the polygenes;

[0130] (b) a plasmid or viral vector; and

[0131] (c) optionally host cells suitable for the propagation of the plasmid or vector

[0132] The primers are conveniently designed to match the needs for producing a single ORF for each polygene and may contain restriction sites for ligation (sequentially or simultaneously) into the plasmid or viral vector and/or the primers may contain sequences for assembling the polygenes and/or insertion into the plasmid or viral vector by homologous recombination (e.g. using the In-Fusion.RTM. system or Red.RTM./ET.RTM. system as described above).

THE FIGURES SHOW

[0133] FIG. 1 shows the nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of a PCR product coding for human TATA-Box-Binding Protein (hTPB) core (hTBPc, c-terminal fragment of the full-length protein truncated at position 159). Positions of RsrII restriction sites (present in the primer sequences) are indicated.

[0134] FIG. 2 shows photographs of agarose gel electrophoretic analyses of in vitro ligation of hTBPc gene segments and subcloning of the mixture. The PCR-amplified hTPBc gene was digested by RsrII and purified (lane 1). Incubation with ligase yields a ladder of concatamers containing 1,2,3 and more genes linked in one ORF each (lane 2, lane 3 is MBI DNA Marker 1 kb ladder). Subcloning of the mixture of the thus-yielded expression constructs containing one polygene each with differing numbers of linked hTBPc genes that can be liberated by restriction digest using RsrII (lanes 4-7). Digestion outside of the inserted polygene evidences 1 (lane 8), 2 (lane 9), 3 (lane 10) and 5 (lane 11) hTBPc genes that yielded a single ORF in each case.

[0135] FIG. 3 shows a schematic representation of the basic transfer vector pSPL underlying preferred transfer vector constructs of the present invention.

[0136] FIG. 4 shows a schematic representation of the basic transfer vector pFL underlying preferred transfer vector constructs of the present invention.

[0137] FIG. 5 shows a schematic representation of the basic transfer vector pKL underlying preferred transfer vector constructs of the present invention.

[0138] FIG. 6 shows a schematic representation of the basic transfer vector pKDM underlying preferred transfer vector constructs of the present invention.

[0139] FIG. 7 shows a schematic representation of the transfer vector construct pFBDO[hTBPc]3.

[0140] FIG. 8 shows the nucleotide sequence of pFBDO[hTBPc]3 (SEQ ID NO: 3).

[0141] FIG. 9 shows a schematic representation of the transfer vector construct pUCDMCSTAF1TBPcTAF2.

[0142] FIG. 10 shows the nucleotide sequence of pUCDMCSTAF1TBPcTAF2 (SEQ ID NO: 4).

[0143] FIG. 11 shows a schematic representation of the transfer vector construct pFBDO[HisTEVTAF6TAF9]his.

[0144] FIG. 12 shows the nucleotide sequence of pFBDO[HisTEVTAF6TAF9]his (SEQ ID NO: 5).

[0145] The present invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

Production of Polygenes and Ligation into Expression Vectors

[0146] The principle of generating polygenes is shown here by using human TATA-Box-Binding protein (hTBP) core (hTBPc, c-terminal fragment of the full-length protein truncated at position 159). The gene encoding hTBPc was amplified by polymerase chain reaction (PCR) using a sense primer annealing to the 5' end of the gene containing an overhang possessing a RsrII restriction site and further encoding an amino acid spacer and a Tobacco-Etch-Virus (TEV) cleavage site. The antisense primer annealed to the 3' terminus of the gene and contained an Rsrll restriction site. RsrII is a restriction enzyme that produces an asymmetric overhang of 3 nucleotides which do not self ligate, therefore, the restriction product is asymmetric and ligation yields a directional product. The PCR product was digested with RsrII and purified. The DNA (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the PCR product are shown in FIG. 1.

[0147] Ligation yielded concatamers of hTBPc as shown in FIG. 2. Subcloning of the in vitro ligation reaction mixture into an appropriate vector yielded expression constructs containing polygenes encoding 1,2,3, and 5 hTBP proteins in a single polyprotein separated by TEV protease cleavage sites. A schematic representation and the nucleotide sequence (SEQ ID NO: 3) of one of the resulting expression vectors (pFBDO[hTBPc]3) are shown in FIGS. 3 and 4, respectively.

Example 2

Generation of Baculoviral Transfer Vectors Containing Polygenes Encoding Subunits of a Human General Transcription Factor

[0148] A polygene was generated encoding a polyprotein comprising human TBP associated factors hTAF1 and hTAF2 in addition to hTBPc inserted into a transfer vector pUCDM (see WO 2005/085456 A1 (PCT/EP2004/013381)) for baculovirus expression, with the genes separated by sequences encoding an amino acid spacer and a TEV protease site. A schematic representation of the resulting construct pUCDMCSTAF1TBPcTAF2 is shown in FIG. 9. The nucleotide sequence of the construct is shown in FIG. 10 (SEQ ID NO: 4). A further construct was generated containing a polygene encoding a polyprotein comprising TEV protease and human TBP associated factors hTAF6 and hTAF9 inserted into the transfer vector pFBDM (see WO 2005/085456 A1 (PCT/EP2004/013381)) for baculovirus expression, with the genes separated by sequences encoding an amino acid spacer and a TEV protease site. A schematic representation of the resulting construct pFDDO[HisTEVTAF6TAF9]his is shown in FIG. 11. The nucleotide sequence of this construct is shown in FIG. 12. (SEQ ID NO: 5)

Example 3

Preparation of Bacmid Constructs, Infection of Insect Cells and Protein Expression

[0149] For the construction of bacmids constructs comprising the above two polygenes, the constructs pUCDMCSTAF1TBPcTAF2 (pUCDM derivative) and pFDDO[HisTEVTAF6TAF9]his (pFBDM derivative) were each introduced into DH10MultiBac.sup.Cre cells as described in Examples 5 (for pUCDMCSTAF1TBPcTAF2; Cre-lox site-specific recombination) and 6 (for pFDDO[HisTEVTAF6TAF9]his; Tn7 transposition) of WO 2005/085456 A1 (PCT/EP2004/013381). If desired, one-step transposition/cre-lox site-specific recombination can be carried DH10MultiBac.sup.Cre cells as described in WO 2005/085456 A1 (PCT/EP2004/013381) as well. Bacmid preparation, infection of insect cells and protein expression was carried out according to established protocols (see, e.g., O'Reilly et al. (1994) "Baculovirus expression vectors. A laboratory manual" Oxford University Press, New York-Oxford; "Bac-to-Bac.TM. Baculovirus Expression Systems Manual" Invitrogen, Life Technologies, Inc., 2000).

[0150] The following Sequence Listing is part of the present description, wherein the sequences are as follows:

[0151] SEQ ID NO: 1 is the nucleotide sequence of the PCR product coding for human TATA-Box-Binding Protein (hTPB) core (hTBPc, c-terminal fragment of the full-length protein truncated at position 159) shown in FIG. 1.

[0152] SEQ ID NO: 2 is the amino acid sequence of the human TATA-Box-Binding Protein core (hTBPc) shown in FIG. 1.

[0153] SEQ ID NO: 3 is the nucleotide sequence of pFBDO[hTBPc]3 shown in FIG. 8.

[0154] SEQ ID NO: 4 is the nucleotide sequence of pUCDMCSTAF1TBPcTAF2 shown in FIG. 10.

[0155] SEQ ID NO: 5 is the nucleotide sequence of pFBDO[HisTEVTAF6TAF9]his shown in FIG. 12.

Sequence CWU 1

1

51607DNAArtificial SequenceSythetic Construct; PCR product coding for hTBPc 1cgaattcctc gagcggtccg gaggtaacgg atccgaaaac ctgtattttc agggttctgg 60gattgtaccg cagctgcaaa atattgtatc cacagtgaat cttggttgta aacttgacct 120aaagaccatt gcacttcgtg cccgaaacgc cgaatataat cccaagcggt ttgctgcggt 180aatcatgagg ataagagagc cacgaaccac ggcactgatt ttcagttctg ggaaaatggt 240gtgcacagga gccaagagtg aagaacagtc cagactggca gcaagaaaat atgctagagt 300tgtacagaag ttgggttttc cagctaagtt cttggacttc aagattcaga acatggtggg 360gagctgtgat gtgaagtttc ctataaggtt agaaggcctt gtgctcaccc accaacaatt 420tagtagttat gagccagagt tatttcctgg tttaatctac agaatgatca aacccagaat 480tgttctcctt atttttgttt ctggaaaagt tgtattaaca ggtgctaaag tcagagcaga 540aatttatgaa gcatttgaaa acatctaccc tattctaaag ggattcagga agacgacgcg 600gtccggc 6072196PRTHomo sapiens 2Arg Ser Gly Gly Asn Gly Ser Glu Asn Leu Tyr Phe Gln Gly Ser Gly 1 5 10 15 Ile Val Pro Gln Leu Gln Asn Ile Val Ser Thr Val Asn Leu Gly Cys 20 25 30 Lys Leu Asp Leu Lys Thr Ile Ala Leu Arg Ala Arg Asn Ala Glu Tyr 35 40 45 Asn Pro Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Arg 50 55 60 Thr Thr Ala Leu Ile Phe Ser Ser Gly Lys Met Val Cys Thr Gly Ala 65 70 75 80 Lys Ser Glu Glu Gln Ser Arg Leu Ala Ala Arg Lys Tyr Ala Arg Val 85 90 95 Val Gln Lys Leu Gly Phe Pro Ala Lys Phe Leu Asp Phe Lys Ile Gln 100 105 110 Asn Met Val Gly Ser Cys Asp Val Lys Phe Pro Ile Arg Leu Glu Gly 115 120 125 Leu Val Leu Thr His Gln Gln Phe Ser Ser Tyr Glu Pro Glu Leu Phe 130 135 140 Pro Gly Leu Ile Tyr Arg Met Ile Lys Pro Arg Ile Val Leu Leu Ile 145 150 155 160 Phe Val Ser Gly Lys Val Val Leu Thr Gly Ala Lys Val Arg Ala Glu 165 170 175 Ile Tyr Glu Ala Phe Glu Asn Ile Tyr Pro Ile Leu Lys Gly Phe Arg 180 185 190 Lys Thr Thr Arg 195 36959DNAArtificial SequenceSynthetic Construct; Plasmid pFBDO[hTBPc]3 3ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1740tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1800tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1860tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1920tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1980ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2040acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2100ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2160gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2220ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2280ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2340taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2400cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2460tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2520agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2580caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2640aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2700cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2760gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2820gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2880ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 2940ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3000cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3060ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3120ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3180atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3240atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3300gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3360cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3420aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3480agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3540gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3600aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3660cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3720aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3780tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3840atagttctag tggttggcct acagctttgt ttaaacaaag ctgtacccgt agtggctatg 3900gcagggcttg ccgccccgac gttggctgcg agccctgggc cttcacccga acttgggggt 3960tggggtgggg aaaaggaaga aacgcgggcg tattggtccc aatggggtct cggtggggta 4020tcgacagagt gccagccctg ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc 4080gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg gttccttccg gtattgtctc 4140cttccgtgtt tcagttagcc tcccccatct cccggtaccg catgctatgc atcagctgct 4200agcaccatgg ctcgagatcc cgggtgatca agtcttcgtc gagtgattgt aaataaaatg 4260taatttacag tatagtattt taattaatat acaaatgatt tgataataat tcttatttaa 4320ctataatata ttgtgttggg ttgaattaaa ggtccgtata ctagtatcga ttcgcgacct 4380actccggaat attaatagat catggagata attaaaatga taaccatctc gcaaataaat 4440aagtatttta ctgttttcgt aacagttttg taataaaaaa acctataaat attccggatt 4500attcataccg tcccaccatc gggcgcggat cctcgagatg ggtaaccatg acaagcgacg 4560atggaaaaag aatttcatag ccgtctcagc agccaaccgc tttaagaaaa tctcatcctc 4620cggggcagct agctggagcc acccgcagtt cgaaaaaggc gccgacgacg acgacgacaa 4680gggctcccat atgtctggga ttgtaccgca gctgcaaaat attgtatcca cagtgaatct 4740tggttgtaaa cttgacctaa agaccattgc acttcgtgcc cgaaacgccg aatataatcc 4800caagcggttt gctgcggtaa tcatgaggat aagagagcca cgaaccacgg cactgatttt 4860cagttctggg aaaatggtgt gcacaggagc caagagtgaa gaacagtcca gactggcagc 4920aagaaaatat gctagagttg tacagaagtt gggttttcca gctaagttct tggacttcaa 4980gattcagaac atggtgggga gctgtgatgt gaagtttcct ataaggttag aaggccttgt 5040gctcacccac caacaattta gtagttatga gccagagtta tttcctggtt taatctacag 5100aatgatcaaa cccagaattg ttctccttat ttttgtttct ggaaaagttg tattaacagg 5160tgctaaagtc agagcagaaa tttatgaagc atttgaaaac atctacccta ttctaaaggg 5220attcaggaag acgacgcggt ccggaggtaa cggatccgaa aacctgtatt ttcagggttc 5280tgggattgta ccgcagctgc aaaatattgt atccacagtg aatcttggtt gtaaacttga 5340cctaaagacc attgcacttc gtgcccgaaa cgccgaatat aatcccaagc ggtttgctgc 5400ggtaatcatg aggataagag agccacgaac cacggcactg attttcagtt ctgggaaaat 5460ggtgtgcaca ggagccaaga gtgaagaaca gtccagactg gcagcaagaa aatatgctag 5520agttgtacag aagttgggtt ttccagctaa gttcttggac ttcaagattc agaacatggt 5580ggggagctgt gatgtgaagt ttcctataag gttagaaggc cttgtgctca cccaccaaca 5640atttagtagt tatgagccag agttatttcc tggtttaatc tacagaatga tcaaacccag 5700aattgttctc cttatttttg tttctggaaa agttgtatta acaggtgcta aagtcagagc 5760agaaatttat gaagcatttg aaaacatcta ccctattcta aagggattca ggaagacgac 5820gcggtccgga ggtaacggat ccgaaaacct gtattttcag ggttctggga ttgtaccgca 5880gctgcaaaat attgtatcca cagtgaatct tggttgtaaa cttgacctaa agaccattgc 5940acttcgtgcc cgaaacgccg aatataatcc caagcggttt gctgcggtaa tcatgaggat 6000aagagagcca cgaaccacgg cactgatttt cagttctggg aaaatggtgt gcacaggagc 6060caagagtgaa gaacagtcca gactggcagc aagaaaatat gctagagttg tacagaagtt 6120gggttttcca gctaagttct tggacttcaa gattcagaac atggtgggga gctgtgatgt 6180gaagtttcct ataaggttag aaggccttgt gctcacccac caacaattta gtagttatga 6240gccagagtta tttcctggtt taatctacag aatgatcaaa cccagaattg ttctccttat 6300ttttgtttct ggaaaagttg tattaacagg tgctaaagtc agagcagaaa tttatgaagc 6360atttgaaaac atctacccta ttctaaaggg attcaggaag acgacgcggt ccggccacca 6420tcatcaccac cattgataag ctagcggccg ctttcgaatc tagagcctgc agtctcgaca 6480agcttgtcga gaagtactag aggatcataa tcagccatac cacatttgta gaggttttac 6540ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg 6600ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 6660atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 6720atgtatctta tcatgtctgg atctgatcac tgcttgagcc taggagatcc gaaccagata 6780agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct 6840acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc 6900cacccctccc agttcccaac gccaactcca tgtgacaaac cgtcatcttc ggctacttt 6959412950DNAArtificial SequenceSynthetic Construct; Plasmid pUCDMSTAF1TBPcTAF2 4aattctgtca gccgttaagt gttcctgtgt cactgaaaat tgctttgaga ggctctaagg 60gcttctcagt gcgttacatc cctggcttgt tgtccacaac cgttaaacct taaaagcttt 120aaaagcctta tatattcttt tttttcttat aaaacttaaa accttagagg ctatttaagt 180tgctgattta tattaatttt attgttcaaa catgagagct tagtacgtga aacatgagag 240cttagtacgt tagccatgag agcttagtac gttagccatg agggtttagt tcgttaaaca 300tgagagctta gtacgttaaa catgagagct tagtacgtga aacatgagag cttagtacgt 360actatcaaca ggttgaactg ctgatcaaca gatcctctac gcggccgcgg taccataact 420tcgtatagca tacattatac gaagttatct ggagtacccg tagtggctat ggcagggctt 480gccgccccga cgttggctgc gagccctggg ccttcacccg aacttggggg ttggggtggg 540gaaaaggaag aaacgcgggc gtattggtcc caatggggtc tcggtggggt atcgacagag 600tgccagccct gggaccgaac cccgcgttta tgaacaaacg acccaacacc cgtgcgtttt 660attctgtctt tttattgccg tcatagcgcg ggttccttcc ggtattgtct ccttccgtgt 720ttcagttagc ctcccccatc tcccggtacc gcatgctatg catcagctgc tagcaccatg 780gctcgagatc ccgggtgatc aagtcttcgt cgagtgattg taaataaaat gtaatttaca 840gtatagtatt ttaattaata tacaaatgat ttgataataa ttcttattta actataatat 900attgtgttgg gttgaattaa aggtccgtat actagtatcg attcgcgacc tactccggaa 960tattaataga tcatggagat aattaaaatg ataaccatct cgcaaataaa taagtatttt 1020actgttttcg taacagtttt gtaataaaaa aacctataaa tattccggat tattcatacc 1080gtcccaccat cgggcgcgga tcctcgagat gggtaaccat gacaagcgac gatggaaaaa 1140gaatttcata gccgtctcag cagccaaccg ctttaagaaa atctcatcct ccggggcagc 1200tagctggagc cacccgcagt tcgaaaaagg cgccgacgac gacgacgaca agggctccca 1260tatgggaccc ggctgcgatt tgctgctgcg gacagcagct accatcactg ctgccgccat 1320catgtcagac acggacagcg acgaagattc cgctggaggc ggcccatttt ctttagcggg 1380tttccttttc ggcaacatca atggagccgg gcagctggag ggggaaagcg tcttggatga 1440tgaatgtaag aagcacttgg caggcttggg ggctttgggg ctgggcagcc tgatcactga 1500actcacggca aatgaagaat tgaccgggac tgacggtgcc ttggtaaatg atgaagggtg 1560ggttaggagt acagaagatg ctgtggacta ttcagacatc aatgaggtgg cagaagatga 1620aagccgaaga taccagcaga cgatggggag cttgcagccc ctttgccact cagattatga 1680tgaagatgac tatgatgctg attgtgaaga cattgattgc aagttgatgc ctcctccacc 1740tccacccccg ggaccaatga agaaggataa ggaccaggat tctattactg gtgagaaagt 1800ggacttcagt agttcctctg actcagaatc tgagatggga cctcaggaag caacacaggc 1860agaatctgaa gatggaaagc tgacccttcc attggctggg attatgcagc atgatgccac 1920caagctgttg ccaagtgtca cagaactttt tccagaattt cgacctggaa aggtgttacg 1980ttttctacgt ctttttggac cagggaagaa tgtcccatct gtttggcgga gtgctcggag 2040aaagaggaag aagaagcacc gtgagctgat acaggaagag cagatccagg aggtggagtg 2100ctcagtagaa tcagaagtca gccagaagtc tttgtggaac tacgactacg ctccaccacc 2160acctccagag cagtgtctct ctgatgatga aatcacgatg atggctcctg tggagtccaa 2220attttcccaa tcaactggag atatagataa agtgacagat accaaaccaa gagtggctga 2280gtggcgttat gggcctgccc gactgtggta tgatatgctg ggtgtccctg aagatggcag 2340tgggtttgac tatggcttca aactgagaaa gacagaacat gaacctgtga taaaatctag 2400aatgatagag gaatttagga aacttgagga aaacaatggc actgatcttc tggctgatga 2460aaacttcctg atggtgacac agctgcattg ggaggatgat atcatctggg atggggagga 2520tgtcaaacac aaagggacaa aacctcagcg tgcaagcctg gcaggctggc ttccttctag 2580catgactagg aatgcgatgg cttacaatgt tcagcaaggt tttgcagcca ctcttgatga 2640tgacaaacct tggtactcca tttttcccat tgacaatgag gatctggtat atggacgctg 2700ggaggacaat atcatttggg atgctcaggc catgccccgg ctgttggaac ctcctgtttt 2760gacacttgat cccaatgatg agaacctcat tttggaaatt cctgatgaga aggaagaggc 2820cacctctaac tccccctcca aggagagtaa gaaggaatca tctctgaaga agagtcgaat 2880tctcttaggg aaaacaggag tcatcaagga ggaaccacag cagaacatgt ctcagccaga 2940agtgaaagat ccatggaatc tctccaatga tgagtattat tatcccaagc aacagggtct 3000tcgaggcacc tttggaggga atattatcca gcattcaatt cctgctgtgg aattacggca 3060gcccttcttt cccacccaca tggggcccat caaactccgg cagttccatc gcccacctct 3120gaaaaagtac tcatttggtg cactttctca gccaggtccc cactcagtcc aacctttgct 3180aaagcacatc aaaaaaaagg ccaagatgag agaacaagag aggcaagctt caggtggtgg 3240agagatgttt tttatgcgca cacctcagga cctcacaggc aaagatggtg atcttattct 3300tgcagaatat agtgaggaaa atggaccctt aatgatgcag gttggcatgg caaccaagat 3360aaagaactat tataaacgga aacctggaaa agatcctgga gcaccagatt gtaaatatgg 3420ggaaactgtt tactgccata catctccttt cctgggttct ctccatcctg gccaattgct 3480gcaagcattt gagaacaacc tttttcgtgc tccaatttat cttcataaga tgccagaaac 3540tgatttcttg atcattcgga caagacaggg ttactatatt cgggaattag tggatatttt 3600tgtggttggc cagcagtgtc ccttgtttga agttcctggg cctaactcca aaagggccaa 3660tacgcatatt cgagactttc tacaggtttt tatttaccgc cttttctgga aaagtaaaga 3720tcggccacgg aggatacgaa tggaagatat aaaaaaagcc tttccttccc attcagaaag 3780cagcatccgg aagaggctaa agctctgcgc tgacttcaaa cgcacaggga tggactcaaa 3840ctggtgggtg cttaagtctg attttcgttt accaacggaa gaagagatca gagctatggt 3900gtcaccagag cagtgctgtg cttattatag catgatagct gcagagcaac gactgaagga 3960tgctggctat ggtgagaaat ccttttttgc tccagaagaa gaaaatgagg aagatttcca 4020gatgaagatt gatgatgaag ttcgcactgc cccttggaac accacaaggg ccttcattgc 4080tgccatgaag ggcaagtgtc tgctagaggt gactggggtg gcagatccca cggggtgtgg 4140tgaaggattc tcctatgtga agattccaaa caaaccaaca cagcagaagg atgataaaga 4200accgcagcca gtgaagaaga cagtgacagg aacagatgca gaccttcgtc gcctttccct 4260gaaaaatgcc aagcaacttc tacgtaaatt tggtgtgcct gaggaagaga ttaaaaagtt 4320gtcccgctgg gaagtgattg atgtggtgcg cacaatgtca acagaacagg ctcgttctgg 4380agaggggccc atgagtaaat ttgcccgtgg atcaaggttt tctgtggctg agcatcaaga 4440gcgttacaaa gaggaatgtc agcgcatctt tgacctacag aacaaggttc tgtcatcaac 4500tgaagtctta tcaactgaca cagacagcag ctcagctgaa gatagtgact ttgaagaaat 4560gggaaagaac attgagaaca tgttgcagaa caagaaaacc agctctcagc tttcacgtga 4620acgggaggaa caggagcgga aggaactaca gcgaatgcta ctggcagcag gctcagcagc 4680atccggaaac aatcacagag atgatgacac agcttccgtg actagcctta actcttctgc 4740cactggacgc tgtctcaaga tttatcgcac gtttcgagat gaagagggga aagagtatgt 4800tcgctgtgag acagtccgaa aaccagctgt cattgatgcc tatgtgcgca tacggactac 4860aaaagatgag gaattcattc gaaaatttgc cctttttgat gaacaacatc gggaagagat 4920gcgaaaagaa cggcggagga ttcaagagca actgaggcgg cttaagagga accaggaaaa 4980ggagaagctt aagggtcctc ctgagaagaa gcccaagaaa atgaaggagc gtcctgacct 5040aaaactgaaa tgtggggcat gtggtgccat tggacacatg aggactaaca aattctgccc 5100cctctattat caaacaaatg cgccaccttc caaccctgtt gccatgacag aagaacagga 5160ggaggagttg gaaaagacag tcattcataa tgataatgaa gaacttatca aggttgaagg 5220gaccaaaatt gtcttgggga aacagctaat tgagagtgcg gatgaggttc gcagaaaatc 5280tctggttctc aagtttccta aacagcagct tcctccaaag aagaaacggc gagttggaac 5340cactgttcac tgtgactatt tgaatagacc tcataagtcc atccaccggc gccgcacaga 5400ccctatggtg acgctgtcgt ccatcttgga gtctatcatc aatgacatga gagatcttcc 5460aaatacatac cctttccaca ctccagtcaa tgcaaaggtt gtaaaggact actacaaaat 5520catcactcgg ccaatggacc tacaaacact ccgcgaaaac gtgcgtaaac gcctctaccc 5580atctcgggaa gagttcagag agcatctgga gctaattgtg aaaaatagtg caacctacaa 5640tgggccaaaa cactcattga ctcagatctc tcaatccatg ctggatctct gtgatgaaaa 5700actcaaagag aaagaagaca aattagctcg cttagagaaa gctatcaacc ccttgctgga 5760tgatgatgac caagtggcgt tttctttcat tctggacaac attgtcaccc agaaaatgat 5820ggcagttcca gattcttggc catttcatca cccagttaat aagaaatttg ttccagatta 5880ttacaaagtg attgtcaatc caatggattt agagaccata cgtaagaaca tctccaagca 5940caagtatcag

agtcgggaga gctttctgga tgatgtaaac cttattctgg ccaacagtgt 6000taagtataat ggacctgaga gtcagtatac taagactgcc caggagattg tgaacgtctg 6060ttaccagaca ttgactgagt atgatgaaca tttgactcaa cttgagaagg atatttgtac 6120tgctaaagaa gcagctttgg aggaagcaga attagaaagc ctggacccaa tgaccccagg 6180gccctacacg cctcagcctc ctgatttgta tgataccaac acatccctca gtatgtctcg 6240agatgcctct gtatttcaag atgagagcaa tatgtctgtc ttggatattc ccagtgccac 6300tccagaaaag caggtaacac aggaaggtga agatggagat ggtgatcttg cagatgaaga 6360ggaaggaact gtacaacagc ctcaagccag tgtcctgtat gaggatttgc ttatgtctga 6420aggagaagat gatgaggaag atgctgggag tgatgaagaa ggagacaatc ctttctctgc 6480tatccagctg agtgaaagtg gaagtgactc tgatgtggga tctggtggaa taagacccaa 6540acaaccccgc atgcttcagg agaacacaag gatggacatg gaaaatgaag aaagcatgat 6600gtcctatgag ggagacggtg gggaggcttc ccatggtttg gaggatagca acatcagtta 6660tgggagctat gaggagcctg atcccaagtc gaacacccaa gacacaagct tcagcagcat 6720cggtgggtat gaggtatcag aggaggaaga agatgaggag gaggaagagc agcgctctgg 6780gccgagcgta ctaagccagg tccacctgtc agaggacgag gaggacagtg aggatttcca 6840ctccattgct ggggacagtg acttggactc tgatgaacgg tccggaggta acggatccga 6900aaacctgtat tttcagggtt ctgggattgt accgcagctg caaaatattg tatccacagt 6960gaatcttggt tgtaaacttg acctaaagac cattgcactt cgtgcccgaa acgccgaata 7020taatcccaag cggtttgctg cggtaatcat gaggataaga gagccacgaa ccacggcact 7080gattttcagt tctgggaaaa tggtgtgcac aggagccaag agtgaagaac agtccagact 7140ggcagcaaga aaatatgcta gagttgtaca gaagttgggt tttccagcta agttcttgga 7200cttcaagatt cagaacatgg tggggagctg tgatgtgaag tttcctataa ggttagaagg 7260ccttgtgctc acccaccaac aatttagtag ttatgagcca gagttatttc ctggtttaat 7320ctacagaatg atcaaaccca gaattgttct ccttattttt gtttctggaa aagttgtatt 7380aacaggtgct aaagtcagag cagaaattta tgaagcattt gaaaacatct accctattct 7440aaagggattc aggaagacga cgcggtccgg aggtaacgga tccgaaaacc tgtattttca 7500gggtgactac aaagacgatg acgataaaaa caggaagaaa ggagacaagg gctttgaaag 7560cccaaggcca tataaattaa cccatcaggt cgtctgcatc aacaacataa atttccagag 7620aaaatctgtt gtgggatttg tggaactgac tatatttccc acagttgcaa acttgaatag 7680aatcaagttg aacagcaaac agtgtagaat ataccgagta aggatcaatg atttagaggc 7740tgcttttatt tataatgacc caaccttgga agtttgtcac agtgaatcaa aacagagaaa 7800cctcaattat ttttccaatg cttatgcagc tgcagttagt gctgtggacc ctgatgcagg 7860aaatggagaa ctttgcatta aggttccatc agagctatgg aaacacgttg atgagttaaa 7920ggtcctgaag atacacatca atttttcttt ggatcagccc aaaggaggtc ttcattttgt 7980ggtacccagt gtagagggaa gtatggcaga gagaggtgct catgttttct cttgtgggta 8040tcaaaattct acaagatttt ggttcccttg tgttgattca tactctgaat tgtgtacatg 8100gaaattagaa tttacagtag atgctgcaat ggttgctgtt tctaatggcg atttggtgga 8160gacagtgtat actcatgata tgaggaagaa aactttccat tatatgctta ccattcctac 8220agcagcgtca aatatctcct tggccattgg accatttgaa atactggtag atccatacat 8280gcatgaggtt actcattttt gtttgcccca acttcttcca ttgctgaaac ataccacatc 8340ataccttcat gaagtctttg aattttatga agaaattctt acatgtcgtt acccatactc 8400ctgttttaag actgtcttca ttgatgaggc ttatgttgaa gtggctgctt atgcttccat 8460gagcattttt agcacaaatc ttttacacag tgccatgatt atagatgaga cacctttgac 8520tagaaggtgt ttagcccaat ccttggccca gcagtttttt ggttgtttca tatctagaat 8580gtcttggtct gatgaatggg tgctgaaggg aatttcaggc tatatctatg gactttggat 8640gaaaaaaact tttggtgtta atgagtaccg ccattggatt aaagaggagc tagacaaaat 8700agtggcatat gaactaaaaa ctggtggggt tttactacat cccatatttg gtggaggaaa 8760agagaaggat aatccggctt cccatctaca cttttcaata aagcatccac atacactgtc 8820ctgggaatac tacactatgt ttcagtgtaa agcccacctt gtgatgagat tgattgaaaa 8880taggatcagt atggaattta tgctacaagt tttcaataaa ctgctaagtc tggctagtac 8940tgcttcatct cagaagttcc agtcacatat gtggagtcag atgttggttt ccacatctgg 9000gtttttgaaa tccatttcaa atgtctctgg caaagatatt cagccgttaa taaagcagtg 9060ggtagatcag agtggagtgg taaaatttta tggaagtttt gcatttaata gaaaacgaaa 9120tgtcttggaa ctggaaataa aacaggacta tacatctcct ggaactcaga aatacgtggg 9180accacttaaa gtgacagtgc aggagttaga tggatccttc aatcatacac tgcaaattga 9240agaaaacagc cttaaacatg atataccctg ccattccaaa agtagaagga ataaaaagaa 9300aaaaatccca ctgatgaatg gagaagaagt tgacatggat ctttctgcaa tggatgctga 9360ttcccctttg ctgtggataa ggatagaccc agatatgtca gtattgagga aggtagaatt 9420tgagcaagct gattttatgt ggcagtatca gctccgctat gagagagatg ttgttgcaca 9480gcaggaatcc attttggctt tggaaaaatt ccctactcca gcatctcggc ttgcactcac 9540tgatatatta gaacaagagc agtgtttcta cagagtaaga atgtcagctt gcttctgtct 9600tgcaaagatt gcaaattcca tggtgagcac atggacagga ccaccagcca tgaagtcact 9660cttcactagg atgttttgtt gtaaaagttg tccaaacatt gtgaaaacaa acaactttat 9720gagctttcaa agttattttc tacagaagac tatgccagtt gcaatggctt tattaagaga 9780tgttcataat ctttgtccta aagaagtctt aacgtttatt ttagacttaa tcaagtacaa 9840tgacaacagg aaaaataagt tttcagataa ctattatcgt gcagaaatga ttgatgccct 9900ggccaactct gttacacctg cagtcagtgt gaataatgaa gttagaactt tggataactt 9960aaatcctgat gtgcgactca ttcttgaaga aatcaccaga tttttgaata tggaaaaact 10020tcttccgagt tacaggcata ccatcactgt cagttgtttg agagccatac gggtacttca 10080gaagaacgga catgtgccaa gtgatccagc tctttttaaa tcttatgctg aatatggcca 10140ctttgtggac attaggatag cagctttgga agcagttgtt gattatacta aagtggacag 10200aagttatgaa gaactgcaat ggctacttaa tatgattcag aatgaccctg taccctatgt 10260aaggcataag attctcaaca tgttgactaa gaacccacca tttactaaga acatggagtc 10320tcccttatgc aatgaagccc tggtagatca actttggaaa cttatgaatt ctggtacttc 10380acatgactgg aggttacggt gtggtgctgt ggacttgtac ttcacacttt ttggcctcag 10440tagaccttcc tgtttaccct tgccagagct tgggttggtt cttaatctaa aggagaaaaa 10500agctgtcttg aatcctacca taattccaga gtcagtagca ggcaaccaag aagctgcaaa 10560taatccaagc agtcacccac agctagttgg atttcagaac cctttttcca gttctcaaga 10620tgaggaggag attgatatgg atactgttca tgatagccag gccttcattt cccatcattt 10680aaacatgctt gaaaggccgt caactccagg gctctcgaag tatcggccag ctagctcccg 10740atctgcttta ataccccagc actcagcagg ctgcgacagc acacccacca caaaacccca 10800gtggagtttg gaacttgcac ggaagggaac aggtaaagaa caagcacctt tggagatgag 10860tatgcatccg gcggcaagcg ctccactctc agtctttact aaggaatcta cagcctccaa 10920acacagtgac caccatcacc accatcacca tgagcacaag aaaaagaaga agaagcataa 10980acataagcac aaacacaagc ataagcatga cagtaaagaa aaggacaagg agcctttcac 11040tttctccagc cctgccagtg gcaggtctat tcgttctcct tccctttcag accggtccgg 11100ccaccatcat caccaccatt gataagctag cggccgcttt cgaatctaga gcctgcagtc 11160tcgacaagct tgtcgagaag tactagagga tcataatcag ccataccaca tttgtagagg 11220ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 11280caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 11340tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 11400tcatcaatgt atcttatcat gtctggatct gatcactgct tgagcctaga agatccggct 11460gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 11520taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata 11580tccggatctg aacaggaggg acagctgata gaaacagaag ccactggagc acctcaaaaa 11640caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt gcgccgaata 11700aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac 11760cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc 11820caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt 11880ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat 11940atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta 12000taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca 12060caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt 12120ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac 12180cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt 12240ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta 12300tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt 12360caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat 12420gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca 12480tgccgtctgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga 12540tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg 12600tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg 12660acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta 12720ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc tgaggccagt 12780ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatcatttat tctgcctccc 12840agagcctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc 12900gcttccttta gcagcccttg cgccctgagt gcttgcggca gcgtgaagct 1295058735DNAArtificial SequenceSynthetic Construct; Plasmid pFBDO[HisTEVTAF6TAF9]his 5ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1740tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1800tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1860tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1920tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1980ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2040acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2100ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2160gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2220ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2280ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2340taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2400cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2460tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2520agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2580caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2640aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2700cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2760gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2820gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2880ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 2940ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3000cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3060ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3120ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3180atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3240atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3300gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3360cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3420aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3480agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3540gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3600aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3660cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3720aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3780tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3840atagttctag tggttggcct acagctttgt ttaaacaaag ctgtacccgt agtggctatg 3900gcagggcttg ccgccccgac gttggctgcg agccctgggc cttcacccga acttgggggt 3960tggggtgggg aaaaggaaga aacgcgggcg tattggtccc aatggggtct cggtggggta 4020tcgacagagt gccagccctg ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc 4080gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg gttccttccg gtattgtctc 4140cttccgtgtt tcagttagcc tcccccatct cccggtaccg catgctatgc atcagctgct 4200agcaccatgg ctcgagatcc cgggtgatca agtcttcgtc gagtgattgt aaataaaatg 4260taatttacag tatagtattt taattaatat acaaatgatt tgataataat tcttatttaa 4320ctataatata ttgtgttggg ttgaattaaa ggtccgtata ctagtatcga ttcgcgacct 4380actccggaat attaatagat catggagata attaaaatga taaccatctc gcaaataaat 4440aagtatttta ctgttttcgt aacagttttg taataaaaaa acctataaat attccggatt 4500attcataccg tcccaccatc gggcgcggat cctcgagatg ggtaaccatc atcatcatca 4560tcacggagaa agcttgttta agggaccacg tgattacaac ccgatatcga gcaccatttg 4620tcatttgacg aatgaatctg atgggcacac aacatcgttg tatggtattg gatttggtcc 4680cttcatcatt acaaacaagc acttgtttag aagaaataat ggaacactgt tggtccaatc 4740actacatggt gtattcaagg tcaagaacac cacgactttg caacaacacc tcattgatgg 4800gagggacatg ataattattc gcatgcctaa ggatttccca ccatttcctc aaaagctgaa 4860atttagagag ccacaaaggg aagagcgcat atgtcttgtg acaaccaact tccaaactaa 4920gagcatgtct agcatggtgt cagacactag ttgcacattc ccttcatctg atggcatatt 4980ctggaagcat tggattcaaa ccaaggatgg gcagtgtggc agtccattag tatcaactag 5040agatgggttc attgttggta tacactcagc atcgaatttc accaacacaa acaattattt 5100cacaagcgtg ccgaaaaact tcatggaatt gttgacaaat caggaggcgc agcagtgggt 5160tagtggttgg cgattaaatg ctgactcagt attgtggggg ggccataaag ttttcatgag 5220caaacctgaa gagccttttc agccagttaa ggaagcgact caactcatga atgaattggt 5280gtactcgcaa ggtggtggtg aaaacctgta cttccagggt aaccacgctg aggagaagaa 5340gctgaagctt agcaacactg tgctgccctc ggagtccatg aaggtggtgg ctgaatccat 5400gggcatcgcc cagattcagg aggagacctg ccagctgcta acggatgagg tcagctaccg 5460catcaaagag atcgcacagg atgccttgaa gttcatgcac atggggaagc ggcagaagct 5520caccaccagt gacattgact acgccttgaa gctaaagaat gtcgagccac tctatggctt 5580ccacgcccag gagttcattc ctttccgctt cgcctctggt gggggccggg agctttactt 5640ctatgaggag aaggaggttg atctgagcga catcatcaat acccctctgc cccgggtgcc 5700cctggacgtc tgcctcaaag ctcattggct gagcatcgag ggctgccagc cagctatccc 5760cgagaacccg cccccagctc ccaaagagca acagaaggct gaagccacag aacccctgaa 5820gtcagccaag ccaggccagg aggaagacgg acccctgaag ggcaaaggtc aaggggccac 5880cacagccgac ggcaaaggga aagagaagaa ggcgccgccc ttgctggagg gggccccctt 5940gcgactgaag ccccggagca tccacgagtt gtctgtggag cagcagctct actacaagga 6000gatcaccgag gcctgcgtgg gctcctgcga ggccaagagg gcggaagccc tgcaaagcat 6060tgccacggac cctggactgt atcagatgct gccacggttc agtaccttta tctcggaggg 6120ggtccgtgtg aacgtggttc agaacaacct ggccctactc atctacctga tgcgtatggt 6180gaaagcgctg atggacaacc ccacgctcta tctagaaaaa tacgtccatg agctgattcc 6240agctgtgatg acctgcatcg tgagcagaca gttgtgcctg cgaccagatg tggacaatca 6300ctgggcactc cgagactttg ctgcccgcct ggtggcccag atctgcaagc attttagcac 6360aaccactaac aacatccagt cccggatcac caagaccttc accaagagct gggtggacga 6420gaagacgccc tggacgactc gttatggctc catcgcaggc ttggctgagc tgggacacga 6480tgttatcaag actctgattc tgccccggct gcagcaggaa ggggagcgga tccgcagtgt 6540gctggacggc cctgtgctga gcaacattga ccggattgga gcagaccatg tgcagagcct 6600cctgctgaaa cactgtgctc ctgttctggc aaagctgcgc ccaccgcctg acaatcagga 6660cgcctatcgg gcagaattcg ggtcccttgg gcccctcctc tgctcccagg tggtcaaggc 6720tcgggcccag gctgctctgc aggctcagca ggtcaacagg accactctga ccatcacgca 6780gccccggccc acgctgaccc tctcgcaggc cccacagcct ggccctcgca cccctggctt 6840gctgaaggtt cctggctcca tcgcacttcc tgtccagaca ctggtgtctg cacgagcggc 6900tgccccacca cagccttccc ctcctccaac caagtttatt gtaatgtcat cgtcctccag 6960cgccccatcc acccagcagg tcctgtccct cagcacctcg gcccccggct caggttccac 7020caccacttcg cccgtcacca ccaccgtccc cagcgtgcag cccatcgtca agttggtctc 7080caccgccacc accgcacccc ccagcactgc tccctctggt cctgggagtg tccagaagta 7140catcgtggtc tcacttcccc caacagggga gggcaaagga ggccccacct cccatccttc 7200tccagttcct cccccggcat cgtccccgtc cccactcagc ggcagtgccc tttgtggggg 7260gaagcaggag gctggggaca gtccccctcc agctccaggg actccaaaag ccaatggctc 7320ccagcccaac tccggctccc ctcagcctgc tccgcggtcc ggtggtggtg gtgaaaacct 7380gtattttcag ggcgagtctg gcaagacggc ttctcccaag agcatgccga aagatgcaca 7440gatgatggca caaatcctga aggatatggg gattacagaa tatgagccaa gagttataaa 7500tcagatgttg gagtttgcct tccgatatgt gaccacaatt ctagatgatg caaaaattta 7560ttcaagccat gctaagaaag ctactgttga tgcagatgat gtgcgattgg caatccagtg 7620ccgcgctgat cagtctttta cctctcctcc cccaagagat tttttattag atattgcaag 7680gcaaagaaat caaacccctt tgccattgat caagccatat tcaggtccaa ggttgccacc 7740tgatagatac tgcttaacag ctccaaacta taggctgaaa tctttacaga aaaaggcatc 7800aacttctgcg ggaagaataa cagtcccgcg gttaagtgtt ggttcagtta ctagcagacc 7860aagtactccc acactaggca caccaacccc acagaccatg tctgtttcaa ctaaagtagg 7920gactcccatg tccctcacag gtcaaaggtt tacagtacag

atgcctactt ctcagtctcc 7980agctgtaaaa gcttcaattc ctgcaacctc agcagttcag aatgttctga ttaatccatc 8040attaatcggg tccaaaaaca ttcttattac cactaatatg atgtcatcac aaaatactgc 8100caatgaatca tcaaatgcat tgaaaagaaa acgtgaagat gatgatgatg acgatgatga 8160tgatgatgac tatgataatc tgcggtccgg ccaccatcat caccaccatt gataagctag 8220cggccgcttt cgaatctaga gcctgcagtc tcgacaagct tgtcgagaag tactagagga 8280tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc 8340tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 8400cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 8460cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct 8520gatcactgct tgagcctagg agatccgaac cagataagtg aaatctagtt ccaaactatt 8580ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca tctattttgt 8640cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt cccaacgcca 8700actccatgtg acaaaccgtc atcttcggct acttt 8735

Claims

1-36. (canceled)

37. A polynucleotide encoding at least two polygenes, wherein each polygene has a single open reading frame (ORF), each polygene comprises at least three genes each coding for a biologically active polypeptide, at least two of the biologically active polypeptides encoded by any genes of the at least two polygenes are of non-viral origin, at least two of the biologically active polypeptides encoded by any genes of the at least two polygenes are each capable of at least transiently interacting with at least one of the other biologically active polypeptides, and the genes constituting each polygene are connected to one another by a sequence coding for at least one self-cleaving peptide, and at least one polygene comprises more than one copy of a gene coding for a biologically active polypeptide.

38. A transgenic non-human animal transformed with the polynucleotide of claim 37.

39. A host cell comprising the polynucleotide of claim 37.