WO2005123765A1

WO2005123765A1 - Process for producing active wnt proteins in prokaryotes

Info

Publication number: WO2005123765A1
Application number: PCT/FI2005/000290
Authority: WO
Inventors: Beatrix Fahnert; Johanna Veijola; Peter Neubauer; Olivier DESTRÉE; Seppo Vainio
Original assignee: Beatrix Fahnert; Johanna Veijola; Peter Neubauer; Destree Olivier; Seppo Vainio
Priority date: 2004-06-17
Filing date: 2005-06-17
Publication date: 2005-12-29
Also published as: FI20040842A0

Abstract

This invention provides a process for producing active Wnt proteins in a bacterial host. The process comprises that a nucleic acid sequence encoding a Wnt protein is expressed in a bacterial host in an oxidative environment in the cytoplasm of a redox mutant strain or the expression product is directed out from the cytoplasm to an oxidative environment capable of retaining the activity of the Wnt protein. The Wnt protein product is recovered from the oxidative environment by recovery methods retaining the activity of the Wnt protein. This invention provides also active Wnt protein produced by the process.

Description

Process for producing active Wnt proteins in prokaryotes

The present invention relates to a process according to the preamble of claim 1 for producing active Wnt proteins and to a Wnt protein product according to the preamble of claim 24 produced by the process.

Background of the Invention

Wnt proteins are a family of secreted, highly-conserved, cysteine-rich glycoproteins inducing an intracellular signalling pathway in eukaryotic cells (Nusse, R. (2003) Development 130, 5297-5305; Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O., and Clevers, H. (1996) Cell 86, 391-399; Behrens, J., von Kries, J. P., Kύhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. (1996) Nature 382, 638-642). Nineteen wnt genes have already been identified in the murine and human genome.

Wnt proteins are beneficial for example for maintaining and differentiating stem cells (e.g. haematopoietic, intestinal epithelium, epidermal and hair follicle stem cells), organ restoration therapy and tissue engineering, because they regulate cell proliferation, differentiation and pattern formation. Even though there is a high demand for active Wnt proteins both in basic research and therapy, the availability of these proteins is limited. There is a need to establish an economic way of producing active Wnt proteins. However, Wnts have molecular features, which make them difficult to produce. They are cysteine- rich, hydrophobic, instable and have basic pi.

Production in eukaryotic cell cultures often results in accumulation of misfolded protein and it has proven difficult to retain activity during the purification of soluble Wnt proteins (Bradley, R. S., and Brown, A. M. C. (1995) Mol. CellBiol 15, 4616-4622).

Only recently Wnts were successfully purified from the growth media of stably transfected eukaryotic cell lines (Willert, K., Brown, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yates, J. R. Ill, and Nusse, R. (2003) Nature 423, 448-452). Yet it has never been successful to produce active Wnt proteins in more economic hosts such as bacterial hosts, yeasts or with a virus system. Quite many patent publications describe Wnt proteins and genes encoding them isolated from various eukaryotic origins, for example WO 01/83543 Al (corresponding US 2003/0175805 Al), US 6297030 Bl, WO 00/12117, WO 01/74856 A2, US 2002/0019029 Al, US 2002/0123103 Al, WO 00/29575, EP 0979870 Al, US 6297030 Bl, US 6,165,751, EP 0887408 Al and US 5,780,291. The patent publications suggest the production of Wnt proteins in a bacterial or other prokaryotic host, but none of them describe the successful production of an active Wnt protein in a prokaryotic host. WO 00/12117 contains a general description of how to express Wnt proteins in E. coli, but the expression system is aimed to express the Wnt protein in the cytoplasm of E. coli and the protein is suggested to be isolated by a denaturing system. By the method of WO 00/12117 it would not be possible to produce active Wnt proteins in the host, since the activity of Wnt protein would be lost in the reducing cytoplasm. Furthermore, the activity of Wnt protein would be lost during the denaturing isolation. Thus a following successful refolding would become necessary. WO 0029575 and US2002/0123103 suggest the production of Wnt proteins through the secretory pathway to the periplasmic space of E. coli, but no guidance is given of how to retain the activity through the whole production process. US 5,780,291 suggests the expression of Wnt-X in E. coli. Wnt-X is produced as insoluble inclusion bodies in the host cells, from where it should be extracted. Inclusion bodies do not contain active protein. Thus a following successful refolding would become necessary.

There is thus a need to establish a production process for active Wnt proteins. In addition, there is a need for a subsequent purification process retaining the activity of the protein.

Summary of the Invention One object of this invention is to provide a process for producing active Wnt proteins.

One further object of this invention is a Wnt protein product produced by the process of this invention.

In this invention it has surprisingly been found that active Wnt proteins can be produced in prokaryotic hosts, in particular in bacterial hosts. The invention is based on the idea of producing Wnt proteins in an oxidative environment or transferring them to an oxidative environment. The cytoplasm of a prokaryotic host is normally a reducing environment. If the host is not a redox mutant the activity of the produced Wnt protein will be lost. An alternative possibility is to translocate the active Wnt product from the reducing cytoplasm into an oxidative environment. The Wnt product may be translocated from the cytoplasm into the periplasm or outside the cell into the medium.

According to a preferred embodiment of this invention a nucleic acid sequence encoding a Wnt protein is expressed in a bacterial host and the expression product is directed from the cytoplasm to an oxidative environment, in particular into the periplasm or into the culture medium, capable of retaining the activity of the Wnt protein. The protein product is recovered from the oxidative environment by recovery methods capable of retaining the activity of the Wnt protein.

According to another preferred embodiment of this invention a nucleic acid sequence encoding a Wnt protein is expressed in a bacterial host under an oxidative environment in the cytoplasm of a redox mutant strain. The protein product is recovered from the cytoplasm of the redox mutant strain by recovery methods capable of retaining the activity of the Wnt protein.

The process for producing active Wnt proteins according to the present invention is mainly characterized by what is stated in claim 1.

A Wnt protein product produced according to the present invention is mainly characterized by what is stated in claim 24.

According to one further preferred embodiment of the invention a nucleic acid sequence encoding a Wnt protein is expressed in a gram-negative bacterial host. The nucleic acid sequence is operably linked into a regulatory region capable of directing the expression product outside from the cytoplasm to the periplasm. The Wnt protein is recovered from the periplasm using recovery methods, which retain the activity of the protein.

According to a highly preferred embodiment of the invention the bacterial host is Escherichia coli.

This invention provides also a method for recovering the active protein product from the periplasmic space. The recovery step comprises one or more of the steps selected from the group comprising extraction, purification, exchange of buffer and concentration. All of the steps are chosen in such a way that the activity of the protein is retained. This invention provides also a method for facilitating the recovery of the active Wnt protein, which comprises that the Wnt protein is tagged in order to facilitate the recovery step.

The present invention fulfils a long-felt need for production of active Wnt protein by a cost-effective manner. Active Wnt proteins can be used for maintaining and differentiating stem cells, for organ restoration therapy and tissue engineering. Active Wnt proteins produced according to the process of the present invention are very useful in basic research and therapy.

Brief description of the drawings

Figure 1 shows the production of soluble internally c-myc-tagged murine Wnt-1 in Escherichia coli

Figure 2 visualises the purification of active internally c-rnyc-tagged murine Wnt-1 from Escherichia coli Figure 3 shows the stabilisation of cytosolic beta-catenin induced by active internally c- myc-tagged murine Wnt-1 from. Escherichia coli

Figure 4 shows that active internally c- yc-tagged murine Wnt-1 from Escherichia coli can lead to axis duplication when injected into the blastocoels of X. laevis embryos

Figure 5 shows that active internally c-myc-tagged murine Wnt-1 from Escherichia coli can induce the appearance of translucent zones in cultivated separated kidney mesenchyme

Figure 6 illustrates the common domain structure of Wnt-proteins as retrieved from the ProDom database. Wnt-proteins from both vertebrates and invertebrates share domains with murine Wnt-1.

Detailed Description of the Invention

Definitions

The term "Wnt proteins" means a family of secreted, highly-conserved, cysteine-rich glycoproteins inducing an intracellular signalling pathway in eukaryotic cells (Nusse, R.

(2003) Development 130, 5297-5305; Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O., and Clevers, H. (1996) Cell 86, 391-399; Behrens, J., von Kries, J. P., Kύhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. (1996) Nature 382, 638-642). Nineteen wnt genes have already been identified in the murine and human genome. Wnt proteins produced according the process of the present invention include members of the Wnt protein family originating from any eukaryote or proteins derived from these through genetic modification of the gene encoding these proteins. Genetic modification comprises for example deletions, substitutions, inversions and insertions, or a combination of these. Wnt proteins from various origins have certain conserved regions with an almost identical amino acid sequence (including important conserved cysteines being very influential in the folding) leading to an overall fold that has certain domains in common. Wnts are ligands exerting their effects through interaction with cell surface receptors of the frizzled family. These frizzled receptors have an extracellular Wnt-binding domain for that purpose. Multiple Wnts can be bound by any given frizzled receptor. This means that the proteins of the Wnt family share domains essential for binding to the receptor and by that for activity. A similar sequence and folding means the production can be approached the same way, because also the mechanism of the folding is shared.

Some of the Wnt-proteins (representing different Wnts from both vertebrates and invertebrates) sharing domains with murine Wnt-1 (Swiss-Prot #P04426) have been retrieved from ProDom for illustration of the common domain structure of the Wnt proteins. ProDom is a comprehensive set of protein domain families automatically generated from the SWISS-PROT and TrEMBL sequence databases, see Figure 6.

The boxes with the same pattern or the same number refer to the same domain. The sometimes different box sizes in the illustration in Figure 6 are due to the display in the data base resulting from variations in the length of the proteins. The similarity would have been even clearer, if the box sizes would have been altered here according to their common size. The original database output has been used to maintain the authenticity of the data.

By "Wnt proteins" are thus meant here proteins, which comprise the amino acid sequence encoded by the original eukaryotic host and also proteins, which have the same function as the original protein, but which comprise an amino acid sequence, which has at least 80 % identity, preferably at least 85 % identity to the amino acid sequence encoded by the original eukaryotic host. More preferably the identity is at least 90 %, still more preferably at least 95 %, most preferably the identity of the amino acid sequence of the invention to the amino acid sequence encoded by the original eukaryotic host is at least 98 %. In this invention as an example of Wnt proteins has been murine Wnt-1. However, Wnt-1 protein from any origin can be produced by the process of this invention. The gene encoding Wnt-1 may be for example from human, mouse, or other mammalian origin. The gene may also originate for example from non-mammalian Xenopus laevis, Caenorhabditis elegans, Brachydanio rerio, Bombyx mori or Ambystoma mexicanum, the DNA sequences of which are available in databases (such as http://www.expasy.ch). This invention includes also the production of a protein comprising the amino acid sequence of murine Wnt-1 and any sequence having at least 30% identity, preferably at least 40 % identity, more preferably at least 50 %, still more preferably at least 60 % identity to the amino acid sequence. The amino acid sequence of the murine Wnt-1 protein can be accessed through the Swiss Protein Database under Swiss-Prot # P04426. More preferably this invention includes the production of a Wnt-1 protein comprising an amino acid sequence having at least 65 % identity, still more preferably at least 75 %, still more preferably at least 85%, still and still more preferably at least 90 %, most preferably the identity of the amino acid sequence of the invention to the published murine amino acid sequence is at least 98 %. The similarities can be determined by Global DNA alignment against a reference molecule (here mWntl, region 1 - 1113), using the Scoring matrix Linear or by Global protein alignment using BLOSUM 62. The term "identity" is used here synonymous to the word "similarity".

The term "oxidative environment" means here the cytoplasm of a redox mutant, which is oxidative, or the term means an oxidative environment outside the cytoplasm, such as the periplasm, or the environment outside the cell wall (culture medium). The formation of disulphide bridges is a prerequisite for activity of Wnt proteins and it requires an oxidative environment. In eukaryotic cells this is the endoplasmic reticulum. In Gram-negative bacteria the Wnt proteins can be translocated to the periplasm or they can even be secreted into the cultivation medium. Secretion into the medium is most common in recombinant protein production with Gram-positive bacteria. An alternative to the translocation out of the reducing bacterial cytoplasm is the use of redox mutant strains having an oxidative cytoplasm. When using redox mutant strains an expression plasmid without a signal sequence for translocation into the periplasm should be constructed. The protein should be extracted after production from the cytoplasm of the host cell instead of the periplasm. By "bacterial host" is in connection of this invention meant gram-positive and gram- negative hosts. Preferred hosts are gram-negative hosts, from these naturally those which are or which have been made not to be harmful for man or environment, which are non- pathogenic and which are safe in use. Examples of Gram-negative hosts are Pseudomonas spec, Serratia spec. , Erwinia spec, Caulobacter crescentus and Enterobacteria, such as Escherichia spec, and Salmonella spec. Most preferred host is Escherichia coli. The preferred Gram-positive host is Bacillus spec.

The use of the mentioned bacteria as production hosts is described quite intensively in literature. Examples of Gram-negative producers are Pseudomonas spec (Penaloza- Vazquez, A., Rangaswamy, V., Ullrich, M., Bailey, A. M., and Bender, C. L. (1996) Use of translational fusions to the maltose-binding protein to produce and purify proteins in Pseudomonas syringae and assess their activity in vivo. Mol Plant Microbe Interact 9, 637- 641.; Lu, S. E., Scholz-Schroeder, B. K., and Gross, D. C. (2002) Construction of pMEKml2, an expression vector for protein production in Pseudomonas syringae. FEMS Microbiol Lett 210, 115-121), Serratia spec, and Erwinia spec. (Rangwala, S. H., Fuchs, R. L., Drahos, D. J., and Olins, P. O. (1991) Broad host-range vector for efficient expression of foreign genes in gram-negative bacteria. Biotechnology (N Y) 9, 477-479), Caulobacter crescentus (Umelo-Njaka, E., Nomellini, J. F., Bingle, W. H., Glasier, L. G., Irvin, R. T., and Smit, J. (2001) Expression and testing of Pseudomonas aeruginosa vaccine candidate proteins prepared with the Caulobacter crescentus S-layer protein expression system. Vaccine 19, 1406-1415) or Enterobacteria such as Salmonella spec (Martin-Gallardo, A., Fleischer, E., Doyle, S. A., Arumugham, R., Collins, P. L., Hildreth, S. W., and Paradiso, P. R. (1993) Expression of the G glycoprotein gene of human respiratory syncytial virus in Salmonella typhimurium. J Gen Virol 1A, 453-458; Jagusztyn-Krynicka, E. K., Clark-Curtiss, J. E., and Curtiss, R. 3rd. (1993) Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins. Infect Immun 61, 1004-1015.; Liljeqvist, S., Haddad, D., Berzins, K., Uhlen, M., and Stahl, S. (1996) A novel expression system for Salmonella typhimurium allowing high production levels, product secretion and efficient recovery. Biochem Biophys Res Commun 218, 356-359.) Shigella spec. (Altboum, Z., Levine, M. M., Galen, J. E., and Barry, E. M. (2003) Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain. Infect Immun 71, 1352-1360.). E. coli has been the most widely used host for recombinant protein production for more than 20 years

(Chan, S. J., Weiss, J., Konrad, M., White, T., Bahl, C, Yu, S. D., Marks, D. and Steiner,

D. F. (1981) Biosynthesis and periplasmic segregation of human proinsulin in Escherichia coli., Proc Nat Acad Sci USA 78, 5401-5405).

The preferred Gram-positive host is Bacillus spec. (Franchi, E., Maisano, F., Testori, S. A., Galli, G., Toma, S., Parente, L., de Ferra, F., and Grandi, G. (1991) A new human growth hormone production process using a recombinant Bacillus subtilis strain, J Biotechnol 18, 41-54).

The bacterial host cells and the genetic constructions used should be chosen in such a way that the Wnt expression product is expressed in oxidative environment or translocated or secreted into an oxidative environment. Oxidative environments in various hosts are described in the following literature.

As described above, the formation of disulphide bridges (a prerequisite for activity) in proteins requires an oxidative environment. In eukaryotic cells the endoplasmic reticulum provides an oxidative environment (Hwang, C, Sinskey, A. J., and Lodish, H. F. (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257, 1496- 1502). When producing disulphide bridged proteins in prokaryotes they have to be directed to a corresponding oxidative environment as well for correct folding. The usual approach is the translocation to the periplasm in Gram-negative bacteria (Raina, S., and Missiakas, D. (1997) Making and breaking disulfide bonds. Annu Rev Microbiol 51, 179-202; Bessette, P. H., Aslund, F., Beckwith, J. and Georgiou, G. (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Nat Acad Sci USA 96, 13703-13708) or even the subsequent secretion into the cultivation medium (Blight, M. A., Chervaux, C, and Holland, I. B. (1994) Protein secretion pathway in Escherichia coli. Curr Opin Biotechnol 5, 468-474; Pines, O., and Inouye, M. (1999) Expression and secretion of proteins in E. coli. Mol Biotechnol 12, 25-34; Cornells, P. (2000) Expressing genes in different Escherichia coli compartments. Curr Opin Biotechnol 11, 450-454.; Lundell, D., Lunn, C, Greenberg, R., Fossetta, J., Narula, S., Kastelein, R., and Van Kimmenade, A. (1990) Exploiting the cell membrane for the production of heterologous proteins in Escherichia coli. Biotechnol Appl Biochem 12, 567-578). Secretion into the medium is most common in recombinant protein production with Gram-positive bacteria (Mosbach, K., Birnbaum, S., Hardy, K., Davies, J., and Bulow, L. (1983) Formation of proinsulin by immobilized Bacillus subtilis. Nature 302, 543-545; Vasantha, N., and Thompson, L. D. (1986) Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences. JBacteriol 165, 837-842).

In case of Escherichia coli as the most widely used host for recombinant protein production, the protein should be directed to the periplasm using a secretory signal sequence. The periplasm provides an oxidising environment. Secreted proteins are also less likely to aggregate in the periplasm (Schein, C. H. (1993) Curr. Opin. Biotechnol. A, 456- 461).

The use of redox mutant strains having an oxidative cytoplasm is an alternative to the translocation out of the reducing bacterial cytoplasm. Mutant strains having an oxidative cytoplasm have been described for example in the following literature: Derman, A. I., Prinz, W. A., Belin, D., and Beckwith, J. (1993) Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262, 1744-1747; Stewart, E. J., Aslund, F., and Beckwith, J. (1998) Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J 17, 5543-5550; Bessette, P. H., Aslund, F., Beckwith, J. and Georgiou, G. (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Nat Acad Sci USA 96, 13703-13708).

By "recovering the protein product from the oxidizing environment by recovery methods retaining the activity of the Wnt protein" is meant that all the recovery steps are chosen in such a way that the activity of the produced Wnt protein is retained during the procedure. The recovery steps comprise one or more steps selected from the group of separation, extraction, purification and exchange of buffer. The recovery step may comprise also concentration. Suitable recovery steps can be chosen from well known recovery steps commonly in use. When choosing a suitable step from various alternatives those methods are preferred that retain the activity of Wnt proteins better. The activity of Wnt proteins can be shown for example by the beta-catenin stabilisation assay or the TOPflash reporter assay. The method depends on the Wnt protein in question, since there are three different signalling cascades. Wntl used here as an example of a Wnt protein starts the canonical signalling cascade. If Wnt proteins are produced in gram-negative bacteria and translocated into the periplasm, the recombinant protein has to be extracted from the periplasm. Different methods are known having a different yield and effect on the proteins. Osmotic shock procedures by means of incubating the cells in a hypertonic solution containing EDTA and then in a hypotonic solution release the components from the periplasmic space (Nossal, N. G., and Heppel, L.E. (1966) J. Biol. Chem. 241,3055-3062). Other extractions are based on detergents, enzymes or organic solvents (Thorstenson, Y. R., Zhang, Y., Olson, P. S., and Mascarenhas, D. (1997) J. Bacteriol. 179, 5333-5339). One preferred method according to this invention is based on osmotic shock procedures.

A preferred way of detecting and isolating proteins is tagging them by introducing a characteristic peptide sequence into the recombinant protein (Terpe, K. (2003) Appl. Microbiol. Biotechnol. 60, 523-533). The Wnt proteins can be tagged with any tag at the N-terminus, C-terminus (in accordance with the usual procedure for designating proteins) or internally. Examples of such tags are the Arg-tag, cellulose-binding domain, c-myc-tag, FLAG-tag, HA-tag, His-tag, S-tag, SBP-tag, Strep-tag and the like. One preferred tag according to the invention is the c-myc-tag (Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616). The purification can then be performed using a resin with an affinity to the tag.

The use of a tag was in this invention exemplified by using an internal tag. The tag sequence can be located to the nucleic acid sequence encoding the amino acids following amino acid 40, preferably amino acid 45, more preferably amino acid 50. In murine Wnt-1 the tag is most preferably located after the amino acid at position 49 in the mature protein of murine Wnt-1.

After purification buffer exchange is usually needed since the protein product cannot be used directly from the purification step. Usually at this time the protein is contained in a solution with a high-salt content or extreme pH value inappropriate for the final use of the Wnt proteins. Also concentration of the produced native protein may be needed and it is often carried out by means of filtration or dialysis (Tsumoto, K., Ejima, D., Kumagai, L, and Arakawa, T. (2003) Protein Expr. Purif. 28, 1-8; Werner, R. G., and Berthold, W. (1988) Arzneimittelforschung 38, 422-428).

Examples of secretion signal sequences used in the process of the present invention include prokaryotic, eukaryotic or viral ones. The signal sequence can be homologous or heterologous to the bacterial host. The signal sequence may originate from any suitable secretion pathway. It can be the signal sequence of a secreted protein, such as OmpA, OmpF, PhoA, PhoE, MBP, beta-lactamase or the like. It may also be the signal sequence of the Wnt protein to be produced or another Wnt protein.

Means for cloning DNA fully encoding the proteins and possibly with tags of the present invention include amplification by PCR using synthetic primers. Cloned protein and possibly tag encoding DNA may be used as such or after digestion with suitable restriction enzymes in accordance with techniques such as that in Molecular Cloning (2nd ed. J Sambrook et al. Cold Spring Harbor Lab. Press (1989). The DNA may have ATG as the translation start codon on the 5' terminal side and TAA, TGA or TAG as the translation stop codon on the 3 ' terminal side.

Expression vectors for the proteins and possibly with tags of the present invention can be manufactured by ligating the DNA fragments downstream of a promoter in a suitable expression vector. Examples of preferred promoters include the ptac, piac, Para, ptet, Ptrp and the like. The preferred promoter is ptac promoter. In addition to the above expression vectors can also include other elements, such as termination sequences, regulative elements (such as lacfi) for instance controlling promoter strength, an fl intergenic region, the origin of replication and selection markers. Examples of selection markers include the ampicillin resistance gene.

The host bacteria can be transformed with vectors containing the nucleic acid sequence encoding the desired Wnt protein and possibly tag sequence. Any Escherichia coli strain can be used. Preferred hosts are those with few auxotrophic markers, little proteolytic activity and suitable for high cell density cultivation, for example RV308 (Morrison, T. G., and Malamy, M. H. (1970) J. Bacteriol. 103, 81-88; Maurer, R., Meyer, B. J., and Ptashne, M. (1980) J. Mol. Biol. 139, 147-161). The cells can be transformed for example in accordance with techniques, such as that described in Molecular Cloning (2nd ed. J Sambrook et al. Cold Spring Harbor Lab. Press (1989).

The bacterial host is preferably cultivated in a rich medium. When culturing Escherichia coli transformants, liquid media are preferred for the culture and should be prepared in such a way as to contain carbon and nitrogen sources, inorganic material and other materials necessary for the growth of such transformants. An example for such a medium is Superbroth (Botstein, K., Lew, K. K., Jarvik, V., and Swanson, C.A. (1975) J. Mol. Biol. 91, 439-462).

In case of Escherichia coli the culture usually takes about 3 to 24 hours, preferably from 3 to 18 hours. The temperature may be about 15 to 43 °C, preferably it is 20 to 37°C. The culture can be aerated, stirred or shaken as needed depending on scale and vessel. The production of the proteins of the present invention, possibly with tags, can be induced at any time during the cultivation and be maintained for any time by adding any inducer in an appropriate concentration depending on the promoter. The induction may take place during 3 to 12, preferably 2 to 6 hours during the cultivation. Preferably the harvest of the cells is carried out immediately after ending the cultivation. An example of an inducer is isopropyl-β-D- thiogalactopyranoside. Other suitable inducers are well known for a person skilled in the art.

The culture is followed by the harvest of the cells by a well known method, such as centrifugation.

When the proteins possibly with tags of the present invention are extracted from the periplasm of the cultured cells a well known method such as Tris-EDTA-Triton extraction, osmotic shock; lysozyme-EDTA treatment, polymyxin B treatment or chloroform treatment and the like can be used, the extract is then obtained by centrifugation, filtration or the like. For example any means of osmotic shock procedure is possible both with self prepared solutions or commercially available kits. When using redox mutant strains an expression plasmid without a signal sequence for translocation into the periplasm should be constructed. The protein should be extracted after production from the cytoplasm of the host cell instead of the periplasm. Methods for the recovery of the protein, such as ultrasonication, are well known for a person skilled in the art.

The proteins possibly with tags contained in the extract obtained in this manner can be purified by a suitable combination of well known methods of isolation and purification. Examples of such methods include methods featuring the use of the degree of dissolution such as solvent precipitation or salting out, methods making use of differences primarily in molecular weight such as dialysis, ultrafiltration, diafiltration, gel filtration, methods making use of differences in charge such as ion exchange chromatography, methods making use of specific affinity including affinity tags such as affinity chromatography, methods making use of hydrophobic differences such as reverse phase HPLC and methods making use of differences in isoelectric point such as isoelectric point electrophoresis and chromatofocussing. Examples for affinity chromatography include such ones based on the c-myc-tag in the proteins of the present invention and anti-c-Myc antibodies contained in the resin. Ultrafiltration can be performed for example by means of commercially available centrifugal filter units.

By using the method of the invention Wnt proteins are obtained which retain their activity. The activity means the ability to induce an intracellular signalling cascade e.g. the canonical Wnt pathway. The activity can thus be shown by methods based on the answers in the signalling chain. The most basic is the beta-catenin stability assay. A description is found on the Wnt-website (http://www.stanford.edu/~rnusse/wntwindow.html) or directly (http ://www. stanford.edu/~rnusse/assays/W3 aPurif .htm#assay) .

According to the description on the website the beta-catenin stabilisation assay is carried out in the following way: Mouse L cells are seeded in 96 well plates, Wnt protein is added (dilution of conditioned medium or dilution of purified protein in complete medium). Cells are incubated for 2 hours in a 37 °C/CO₂ incubator. Medium is aspirated. Cells are washed once with PBS. PBS is aspirated. Cells are lysed by adding 30 μl lysis buffer (1% Triton X-100, 150mM NaCl, 50mM Tris-HCl, pH8). The lysate is added to Laemmli buffer and boiled for 5 minutes. 20 μl of each sample is resolved by SDS-10% PAGE. Proteins are transferred to nitrocellulose and blotted with a mouse anti-beta-catenin antibody.

The proteins of the present invention, possibly with tags, can be used in the usual manner. The resulting preparation is safe and has low toxicity.

Examples are given below to illustrate the present invention in further detail, but the scope of the present invention is not limited by these examples. Genetic manipulation involving the use of Escherichia coli was based on the methods described in Molecular Cloning (2nd ed. J Sambrook et al. Cold Spring Harbor Lab. Press (1989). Examples Example 1 Construction of internally c-myc-tagged murine Wnt-1 expressing Escherichia coli strain with the protein translocated into the periplasm using the signal sequence of the Escherichia coli OmpA The structural gene of mature Mwnt-1 with a c-myc-tag situated after the 49^th amino acid was amplified by PCR from pgem4int-lmycpolyA (McMahon, A. P., and Moon, R. T. (1989) Cell 58, 1075-1084) using a primer 1 (5'-

AGCGCTAGCGCCAACAGTAGTGGCCGATG-3' having an Nhel cleavage site upstream of the structural gene and a primer 2 (5'- TCAAAGCTTGAATTCGAGCTCTCATAGACACTCGTGCAGAAC-3') having a Hindlll cleavage site adjacent downstream of a stop codon. The gene thus amplified by PCR was digested with Nhel and Hindlll and ligated using T4 DNA ligase to the vector pBF005 (Fahnert, B. (2001) Rekombinαntes humαnes BMP-2 αus Escherichia coli - Strategien zur Expression und Funktionalisierung, Doctorate Thesis, Friedrich-Schiller- University Jena, Germany) digested with the same enzymes to prepare pBFompAwntlmyc. This was introduced to Escherichia coli RV308 and transformants were selected using ampiciUin resistance. Transformants having pBFompAwntlmyc were cultured. The plasmid encodes mwnt-lmyc precursor gene (with the corresponding signal sequence) with an lpp termination sequence under control of ptac additionally regulated by laclq under p_laoι control, an fl intergenic region, an ampiciUin resistance marker and the ColEl origin.

Example 2 Construction of internally c-myc-tagged murine Wnt-1 expressing Escherichia coli strain with the protein translocated into the periplasm using its own signal sequence

The structural gene of the full-length Mwnt-1 with a c-myc-tag situated after the 49^th amino acid of the mature protein was amplified by PCR from pgem4int-lmycpolyA (McMahon, A. P., and Moon, R. T. (1989) Cell 58, 1075-1084) using a primer 1 (5'- CCTAGGCCTATGGGGCTCTGGGCGCTG-3' having a Stul cleavage site and start codon and a primer 2 (5'-

TCAAAGCTTGAATTCGAGCTCTCATAGACACTCGTGCAGAAC-3') having a Hindlll cleavage site adjacent downstream of a stop codon. The gene thus amplified by PCR was digested with Stul and Hindlll and ligated using T4 DNA ligase to the vector pBF005 (Fahnert, B. (2001) Rekombinαntes humαnes BMP-2 αus Escherichia coli - Strategien zur Expression und Funktionalisierung, Doctorate Thesis, Friedrich-Schiller- University Jena, Germany) digested with the same enzymes to prepare pBFsswntlmyc c.

This was introduced to Escherichia coli RV308 and transformants were selected using ampiciUin resistance. Transformants having pBFsswntlmyc were cultured. The plasmid encodes the sequence of the mature mwnt-1 myc gene behind the signal sequence of the E. coli ompA gene with an lpp termination sequence under control of ptac additionally regulated by laclq under p_lacι control, an fl intergenic region, an ampiciUin resistance marker and the ColEl origin.

Example 3 Culture of Escherichia coli RV308 harbouring pBFompAwntlmyc or pBFsswntlmyc, respectively The Escherichia coli strain RV308 harbouring either pBFsswntlmyc or pBFompAwntlmyc was grown in 15 ml Superbroth (3.5 % Tryptone, 2.0 % yeast extract, 0.5 % NaCl, pH 7.5; Botstein, K., Lew, K. K., Jarvik, V., and Swanson, C.A. (1975) J. Mol. Biol. 91, 439-462) supplemented with 100 μg ml^"1 ampiciUin at 37 °C over night. This pre-culture was used to inoculate the main culture in the same medium to a starting optical density of 0.1 at 550 nm. The main culture was performed at 37 °C, protein production induced with 1 mM isopropyl-β-D-thiogalactopyranoside when an optical density of 0.5 at 550 nm was reached and cultivation was continued for 4 h. Then the cells were centrifuged at 8,500xg for 5 min at 4 °C.

Figure 1 is the developed film of a chemiluminescence detection of the binding of anti- mouse-horseradish-peroxidase to anti-c-Myc to the c-myc- ag in c-myc-tagged murine Wnt-1 produced in A) Escherichia coli RV308 cells harbouring pBFsswntlmyc and B) Escherichia coli RV308 cells harbouring pBFompAwntlmyc grown at 37 °C in Superbroth with a protein production induced with IPTG for 4h after ultrasonication of the cells, centrifugation of the whole cell extract (C) to separate soluble (S) and insoluble (IS) fraction, electrophoresis of the proteins in an SDS-polyacrylamide gel and Western blotting giving the results as described above. The migration positions of molecular weight markers are shown on the left. The target protein is indicated by an arrow. As shown in Figure 1 internally c-røyc-tagged murine Wnt-1 can be produced soluble in Escherichia coli. Example 4 Purification of active internally c-myc-tagged murine Wnt-1 from Escherichia coliRV308 cells harbouring pBFompAwntlmyc or pBFsswntlmyc, respectively 0.9 g cells obtained in Example 3 were resuspended in 1.8 ml of PeriPreps Periplasting

Buffer (PeriPreps™ Periplasting Kit, by Epicentre) and incubated for 5 min at room temperature. 2.25 ml of ice-cold purified water of the same kit were added, everything kept on ice for 10 min and then centrifuged for 15 min at 8,000xg and 4 °C. The supernatant was centrifuged again at the same parameters. For the purification 1 ml of anti-c-Myc Agarose Conjugate (by Sigma-Aldrich) was used according to the manufacturer with some modifications. The slurry was packed in Polyprep Chromatography Columns (by Bio-Rad Laboratories) and equilibrated with 3 x 5 ml of PBS (0.137 M NaCl, 2.68 mM KC1, 10 mM Na₂HPO_4> 2 mM KH₂PO₄, pH 7.4). After loading the sample the column was washed with 3 x 5 ml of PBS. The elution was performed with 9 x 1 ml of 0.1 M NH₄OH (pH 11.5). The pH of the fractions had to be neutralised. 10 μl of 1 M acetic acid was needed for the first and 40 μl for the other fractions. The acid had already been provided in the tubes for collecting the fractions.

Centricon YM-50 Centrifugal Filter Units (by Millipore) were used for buffer exchange of the eluted fractions 1 to 3 and concentration. The units were equilibrated with 2 ml PBS for 15 min at 4 °C and 5.000xg. Then the devices were inverted and centrifuged for 2 min at 4 °C and 500xg. Fractions 1 to 3 were consecutively load and centrifuged for 10 min at 4 °C and 5.000xg. Thereafter the filter units were washed 5 times with 2 ml PBS and each time centrifuged for 15 min at 4 °C and 5.000xg. For harvesting the purified protein in a clean vessel the device was inverted and centrifuged for 2 min at 4 °C and 300xg.

As shown in Figure 2 the bound protein could be eluted mainly in the second and third fraction and accumulated exclusively in the retentate after ultrafiltration.

Figure 2 is the developed film of a chenn uminescence detection of the binding of anti- mouse-horseradish-peroxidase to anti-c-Myc to the c-myc-tag in c-myc-tagged murine Wnt-1 produced in Escherichia coli RV308 cells harbouring pBFsswntlmyc after harvesting the cells grown at 37 °C in Superbroth producing the recombinant protein induced with IPTG for 4 h, loading the periplasmic extract (L) on anti-c-Myc conjugated agarose, collecting the flowthrough (FT), washing fractions (W) and eluted fractions 1 to 4 (E1-E4), collecting the retentate (R) after buffer exchange using YM-50 centrifugal units, sampling the resin (Col), electrophoresis of the proteins in an SDS-polyacrylamide gel and Western blotting giving the results as described above. The migration positions of molecular weight markers are shown on the left. The target protein is indicated by an arrow. The bound protein could be eluted mainly in the second and third fraction. Due to different processing volumes the concentrations of the samples are not the same.

Figure 2 shows that active internally c-myc-tagged murine Wnt-1 can be purified from Escherichia coli. Example 5 Stabilisation of beta-catenin by active internally c-myc-tagged murine Wnt-1 from Escherichia coli

MDCK cells (130,000 in 500 μl of medium per well) were seeded in 24 weU plates the day before the assay. At a confluency of 80 to 90 % the commonly used cultivation medium was changed against 500 μl of the same medium plus 100 U ml^"1 penicillin, 100 μg ml¹ streptomycin sulphate and 10 μl of the purified samples of recombinant protein to be tested (final concentration 1:50; 1:250; 1:500; 1:1000) or the negative control (plasmid without the wnt-1 gene used for the whole production process) or the positive control (20 mM LiCl) were added in duplicate. After the plates had been incubated for 2.5 hours the cells were harvested and resuspended in lysis buffer (150 mM NaCl; 50 mM Tris, pH=8.0; 1 % Triton X-114, Sigma). Then the samples were subjected to immunoblotting. Anti-active- beta-catenin antibodies (clone 8E7, mouse monoclonal, Upstate) were used as the first antibody and anti-mouse-horseradish-peroxidase as the secondary according to the manufacturer.

As shown in Figure 3 by the negative control β-catenin was not stabilised but by LiCl. The stabilisation of beta-catenin by the recombinant internally c-røyc-tagged murine Wnt-1 was concentration-dependent. The 1:50 and 1:250 dilutions seemed to have a comparable effect. In case of higher dilutions there was less beta-catenin detected.

Figure 3 is the developed film of a chemiluminescence detection of the binding of anti- mouse-horseradish-peroxidase to anti-active-beta-catenin antibodies from mouse to cytosolic beta-catenin from MDCK cells. After adding purified samples of protein translocated using the host ompA signal sequence and protein translocated due to its own signal sequence (final concentrations from 1:50 to 1:1000), the negative control (nc, plasmid without the wnt-1 gene used for the whole production process) or the positive control (pc, 20 mM LiCl) in duplicate to the culture medium of MDCK cells and incubating the cells were harvested. The samples were separated in electrophoresis in an SDS-polyacrylamide gel and analysed by Western blotting giving the results as described above. The migration position of the molecular weight marker is shown on the left. The beta-catenin is indicated by an arrow. The stabilisation of beta-catenin by the recombinant Wnt was seen to be concentration-dependent indicating activity of the recombinant mWnt- 1. Figure 3 shows that active internally c-myc-tagged murine Wnt-1 from Escherichia coli can induce stabilisation of cytosolic beta-catenin.

Example 6 Induction of axis duplication by injection of active internally c-myc-tagged murine Wnt-1 into the blastocoel ofX. laevis embryos

X. laevis embryos were obtained by in vitro fertilisation and raised in 25 % modified Marc's Ringer at 18 °C. Embryos at stage 7-8, were injected with 50 nl of the purified c- myc-tagged murine Wnt-1 protein into the blastocoel. The embryos were kept for 6 h in 25% modified Marc's Ringer with 3% Ficoll (Amersham Biosciences) at 18 °C, then were raised in medium without Ficoll at 18 °C, and were fixed at tail bud stage in 100 mM Mops, pH 7.4, 2 mM EGTA, 1 mM MgSO4, 4% paraformaldehyde before being analysed for the formation of a secondary axis. The injection of 50 nl of a solution containing purified recombinant protein (product of pBFompAwntlmyc) resulted in formation of secondary axes in 18.8% (n = 16) of the embryos, whereas the product of pBFsswntlmyc resulted in 42% (n = 19) in the formation of secondary axes. In the embryos showing two axes neither the ectopic nor the endogenous axis contained anterior structures such as eyes and cement glands (Fig. 4, middle and bottom, arrows). Thus, the recombinant Mwnt-1 injected into the blastocoel affected both early and late canonical Wnt signalling resulting in a combination of induction of a secondary axis and inhibition of head formation. The injected protein obviously remained active for a certain time because the endogenous axis did not form a proper head leading to defects similar to those seen after wnt-1 -DNA injection (not shown). Injection of 50 nl of the negative control did not induce any ectopic body axis and embryos showed normal development of the eyes and cement gland (e.g.) (Fig. 4, top).

Figure 4 is the picture of the raised and fixed X. laevis embryos after injecting a solution containing purified c-myc-tagged murine Wnt-1 (middle and bottom) protein or the negative control (top) into the blastocoel at stage 7-8. As opposed to the negative control the injected protein was active for a certain time giving the results (formation of secondary axes without anterior structures such as eyes and cement glands) as described above. Figure 4 shows that active internally c-myc-tagged murine Wnt-1 from Escherichia coli can lead to axis duplication when injected into the blastocoels of X. laevis embryos.

Example 7. Induction of early stage kidney tubules by applying active recombinant internally c-myc-tagged murine Wnt-1 protein on separated embryonic kidney mesenchymes

The embryonic kidney was separated from 11.5 day old mouse embryos by microsurgical operation. The spinal cord was detached from the caudal embryonic region and the two ventrally located urogenital systems were separated in Dulbecco's buffered solution, pH 7.4. The metanephric kidney was further isolated from the rest of the mesonephros. The separated kidney rudiments were incubated 30-60 seconds in ice-cold Tyrode's solution containing 2.25 % pancreatin and 0.75 % trypsin. Then the ureteric bud (the natural tubule inducer) was mechanically separated from the predetermined metanephric mesenchyme after 10 min incubation at room temperature in DMEM with 10 % fetal calf serum and antibiotics. Separated kidney mesenchymes were placed on sliced Nuclepore filters with a pore size of 0.6 μm. Purified c-myc-tagged Wnt-1 was added directly to the culture medium in dilutions of 1:10 and 1:30 and incubation at 5 % CO₂ in humidified air at 37 °C for 23 hours. When the mesenchyme was cultured uninduced in control media without myc-tagged Wnt-1 neither a translucent zone nor tubules appeared during the first 23 h of culture serving as a negative control (Fig. 5, top). A heterologous inducer such as a piece of embryonic spinal cord or lithium chloride (Fig. 5, middle) triggered the appearance of translucent zones in the separated kidney mesenchyme during the first 23 h of culture marking the induction of kidney tubulogenesis. Recombinant myc-tagged Wnt-1 was also sufficient to induce the early steps of kidney tubulogenesis during the first 23 h of mesenchymal organ culture as judged by the appearance of the translucent area in the explant (Fig. 5, bottom).

Figure 5 is the picture of separated kidney mesenchyme cultured with or without inducers. In the negative control neither translucent areas nor tubules appeared in the mesenchyme. Lithium chloride (positive control) known as an inducer of early steps of kidney tubules induced a translucent zone in the separated kidney mesenchyme. As opposed to the negative control and in accordance with the positive control myc-tagged Wnt-1 (in 1:30 dilution) induced a translucent zone in the centre of the explant thus implying inductive property. This indicates that the recombinant protein was active when assayed as described above. Figure 5 shows that active internally c-myc-tagged murine Wnt-1 from Escherichia coli can induce the appearance of translucent zones in cultivated separated kidney mesenchyme.

Claims

1. A process for producing active Wnt proteins, which comprises:

- expressing a nucleic acid sequence encoding a Wnt protein in a bacterial host in an oxidative environment in the cytoplasm of a redox mutant strain, or directing the expression product out from the cytoplasm to an oxidative environment capable of retaining the activity of the Wnt protein, — recovering the protein product from the oxidative environment by recovery methods retaining the activity of the Wnt protein.

2. The process according to claim 1, wherein the oxidative environment is the cytoplasm of a redox mutant strain, the periplasm of a gram-negative bacterium, or the culture medium of the bacterial host.

3. The process according to claim 1 or 2, wherein the bacterial host is a gram- negative bacterium.

4. The process according to any one of claims 1 to 3, wherein the bacterial host is selected from the group comprising Enterobacteria, preferably Escherichia spec, or Salmonella spec, or is selected from the group comprising Pseudomonas spec, Serratia spec, Erwinia spec, and Caulobacter crescentus.

5. The process according to any one of claims 1 to 4, wherein the bacterial host is Escherichia coli.

6. The process according to any one of the preceding claims, wherein the nucleic acid sequence is operably linked to a regulatory region capable of directing the expression product from the cytoplasm into the periplasm or to the culture medium.

7. The process accordmg to any one of the preceding claims, wherein the regulatory region directs the protein to the periplasmic space.

8. The process according to claim 1 or 2, wherein the bacterial host is a gram- positive bacterium.

9. The process according to claim 8, wherein the bacterial host belongs to Bacillus spec.

10. The process according to any one of the preceding claims, wherein the regulatory region comprises a secretion signal sequence.

11. The process according to any one of the preceding claims, wherein the recovery comprises one or more steps selected from the group of extraction, purification, exchange of buffer and optionally concentration of the protein.

12. The process according to any one of the preceding claims, wherein the Wnt protein is tagged.

13. The process according to any one of the preceding claims, wherein the Wnt protein is tagged with a c-myc-tag.

14. The process according to any one of the preceding claims, wherein the secretion signal sequence is the secretion signal sequence of a secretory protein of the bacterial host.

15. The process accordmg to any one of the preceding claims, wherein the secretion signal sequence is the secretion signal sequence of a secretory protein of Escherichia coli.

16. The process according to any one of the preceding claims, wherein the secretion signal sequence is the secretion signal sequence of ompA gene.

17. The process according to any one of claims 1 to 13, wherein the secretion signal sequence is the secretion signal sequence of a Wnt protein.

18. The process according to any one of the preceding claims, wherein the protein expression is controlled by an inducible promoter.

19. The process according to claim 18, wherein the protein expression is controlled by ptac promoter.

20. The process according to any one of the preceding claims, wherein the bacterial host is cultivated in a rich medium.

21. The process according to any one of the preceding claims, wherein the Wnt protein is recovered from the periplasm of the bacterial host by an osmotic shock.

22. The process according to any one of the preceding claims, wherein the Wnt protein is purified by chromatography.

23. The process according to any one of the preceding claims, wherein the Wnt protein is a protein comprising the amino acid sequence of murine mature Wnt- 1 or a sequence having at least 30% identity to murine mature Wnt-1 protein with or without its signal sequence.

24. A Wnt protein product produced by the process according to any one of claims 1 to 23, wherein the protein is capable of inducing intracellular signalling.

25. The Wnt protein product according to claim 24, wherein the protein is in non- glycosylated form.

26. Use of the oxidative environment of the cytoplasm of a redox mutant strain or the periplasmic space of a gram-negative bacterium for producing active Wnt proteins.