RECOMBINANT VACCINE AGAINST HELMINTHS IN PICHIA PASTORIS AND METHODS FOR PRODUCING AND PURIFYING PROTEINS FOR USE AS VACCINES AGAINST HELMINTHS

Abstract

The present invention is related to the recombinant production of proteins by using a synthetic gene for high protein expression in Pichia pastoris. More specifically, the invention describes the production of Sm14 Schistosoma mansoni recombinant protein, where a synthetic gene was created to promote high expression of such protein, a gene which was cloned under control of two types of Pichia pastoris promoters: methanol-inducible promoter (AOXI) and constituent promoter (GAP). With these constructions, Pichia pastoris strains were genetically manipulated to efficiently produce vaccine antigen Sm14. The processes to produce and purify this protein from P. pastoris cells, which can be escalated for their industrial production, were also improved.

Claims

1-6. (canceled)

7. A process of producing a recombinant protein Sm14 of Schistosoma mansoni in Pichia pastoris, comprising the steps of: (a) synthesizing the gene defined by SEQ ID NO:3; (b) cloning of the synthesized gene of SEQ ID NO: 3 in vector pPIC9K by BamHI site and reconstituting the Kozak sequence of gene AOX1 before the start codon of protein Sm14, for the expression of protein Sm14 in its intracellular form, induced by methanol; (c) replacing promoter AOX1 by promoter gene GAP in plasmid pPIC9K-Sm14-MV, with the SacI and BamHI sites, for the constituent expression of protein Sm14; (d) transforming P. pastoris cells with plasmids pPIC9K-Sm14-MV and pGAP9K-Sm14-MV, and selecting recombinant clones with multiple copies.

8. A process of producing a recombinant protein Sm14 of Schistosoma mansoni expressed in Pichia pastoris, comprising the steps of: (a) Performing the lysis of P. pastoris cells; (b) clarifying the lysate obtained in stage (a) in order to obtain a clarified lysate; (c) Conditioning the clarified lysate of step (b) through tangential filtration in preparation for performing ion exchange chromatography; (d) loading the clarified lysate in an anion-exchange resin and after loading the protein, the protein is eluted by pH changes in the column; (d) Separating the contaminant proteins from the recombinant protein by gel-filtration, wherein the protein is coded by sequence SEQ ID NO: 3 and is expressed in Pichia pastoris.

9. A purification process according to claim 8 wherein the process is used in the purification of binding proteins of fatty acids of other parasites with physical-chemical properties that are similar to protein Sm14.

10. A purification process according to claim 8 wherein the process is used in the purification of protein type-3 FABP of Fasciola hepatica

11. A vaccine composition comprising a protein expressed in Pichia pastoris obtained by the process disclosed in claim 8.

12. A therapeutic composition comprising a protein expressed in Pichia pastoris obtained by the process disclosed in claim 8.

13. A diagnostic agent comprising a protein expressed in Pichia pastoris obtained by the process disclosed in claim 8.

14. A vaccine for the prevention and/or treatment of infections caused by helminths, schistosomiasis, fasciolosis, echinococcosis, combinations thereof, and other helminth diseases of human and veterinary relevance.

15. A Synthetic gene characterized by being obtained according to the process of claim 7.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 shows the strategy for building the pPIC9K-Sm14-MV and pGAP9K-Sm14-MV plasmids.

[0012] FIG. 2 shows the induction of protein Sm14 expression in P. pastoris GS115/pPIC9K-Sm14-MV.

[0013] FIG. 3 shows protein Sm14 expression in P. pastoris GSI15/pGAP9K-Sm14-MV.

[0014] FIG. 4 shows the result of the induction of protein Sm14 expression in P. pastoris GS115/pPIC9K-Sm14-MV culture in fermenter.

[0015] FIG. 5 shows the western blot analysis of purified protein of P. pastoris.

[0016] FIG. 6 shows the circular dichroism spectrum of P. pastoris purified protein Sm14.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The main purpose of the invention which is to produce a recombinant vaccine against helminths, can be achieved by producing recombinant proteins using a synthetic gene for high protein expression in Pichia pastoris. According to the invention a synthetic gene was created to promote high expression of protein Sm14, and with such gene the Pichia pastoris strain was obtained and genetically manipulated to effectively produce a vaccine. This invention further contemplates this protein production and purification processes from P. pastoris cells; which can be escalated for industrial production.

[0018] A Pichia pastoris is an efficient host to express and secrete heterologous proteins. The main promoter used for expression in this system is the strongly and firmly regulated promoter from the alcohol oxidase 1 gene (AOX1), inducible by methanol. However, this invention describes a system using an alternative promoter to prevent the use of methanol. The promoter of enzyme glyceraldehyde 3 phosphate dehydrogenase (pGAP) gene was used in the present invention for the constituent expression of the heterologous protein, once this system is more appropriate for large scale production because it eliminates the risk and costs associated with the storage and supply of large volume of methanol.

[0019] The invention will now be described through its best execution process. The technical matter of patent application PI 1005855-9 which reflects the closest state-of-the-art of the present invention will be attached here for reference purposes.

1. Obtainment of Pichia pastoris Recombinant Strain
1.1 Synthetic Gene for Sm14 Expression in P. pastoris:

[0020] First a gene was designed and synthesized containing optimized codons to obtain maximum expression of protein Sm14 in P. pastoris. In the present case protein Sm14-MV was used; however any form of protein Sm14 can be used.

[0021] There is evidence in the literature about the differential use of codons between proteins expressed in low amount and high amount, in the same organism (Roymondal and Sahoo, 2009). However, the codon use tables available in databases (for example: (www.kazusa.or.jp/codon) contain data from all body proteins, without taking the gene expression level into account. For this reason, in order the gene design, a codon use table was first elaborated based on data about sequences that codify recombinant proteins expressed over 1 gram per culture Liter in P. pastoris (see Table 1), as well as the sequence for AOX1 protein (which represents 30% of total P. pastoris protein, after induction with methanol).

TABLE-US-00001 TABLE 1 List of high-expression recombinant proteins in P. pastoris. Expressed protein (mg/L) Reference Hydroxynitrile 22000 Hasslacher, M. et al. (1997) Protein lyase Expr. Purif. 11: 61-71 Mouse gelatin 14800 Werten, M. W. et al. (1999) Yeast 15: 1087-1096 Tetanus toxin 12000 Clare, J. J. et al. (1991) Bio/Technology fragment C 9: 455-460 Human tumor 10000 Sreekrishna, K. et al. (1989) Biochemistry necrosis factor 28: 4117-4125 -amylase 2500 Paifer, E. et al. (1994) Yeast 10: 1415-1419 T2A peroxidase 2470 Thomas, L. et al. (1998) Can. J. Microbiol. 44: 364-372 Catalase L 2300 Calera, J. A. et al. (1997) Infect. Immun. 65: 4718-4724 Hirudin 1500 Rosenfeld, S. A. et al. (1996) Protein Expr. Purif. 8: 476-482.

[0022] For gene design, protein Sm14-MV sequence, which has a valine residue at position 62 has been chosenin place of cystein, which makes it more stable (Ramos et al., 2009); represented here as SEQ ID NO:1.

TABLE-US-00002 SEQIDNO:1 MSSFLGKWKLSESHNFDAVMSKLGVSWATRQIGNTVTPTV TFTMDGDKMTMLTESTFKNLSVTFKFGEEFDEKTSDGRNV KSVVEKNSESKLTQTQVDPKNTTVIVREVDGDTMKTTVTV GDVTAIRNYKRLS

[0023] After the first selection of codons according to the table created with protein data from Table 1, the clearance of sequences which resulted in transcription termination sites (ATTTA), splicing cryptic donors and receptors (MAGGTRAGT and YYYNTAGC, respectively) and repetitive sequences (cut off sites for restriction enzymes BamHI and EcoRI) was conducted. SEQ ID NO:2 shows the sequence designed to express protein Sm14-MV in Pichia pastoris.

TABLE-US-00003 SEQIDNO:2 1 ATGTCTTCTTTCTTGGGTAAGTGGAAGTTGTCTGAATCTCACAACTTCGA 51 CGCTGTTATGTCTAAGTTGGGTGTTTCTTGGGCTACCAGACAAATTGGTA 101 ACACCGTTACTCCAACCGTTACCTTCACCATGGACGGTGACAAGATGACT 151 ATGTTGACCGAGTCTACCTTCAAGAACTTGTCTGTTACTTTCAAGTTCGG 201 TGAAGAGTTCGACGAAAAGACTTCTGACGGTAGAAACGTTAAGTCTGTTG 251 TTGAAAAGAACTCTGAATCTAAGTTGACTCAAACTCAAGTTGACCCAAAG 301 AACACTACCGTTATCGTTAGAGAAGTTGACGGTGACACTATGAAGACTAC 351 TGTTACCGTTGGTGACGTTACCGCTATCAGAAACTACAAGAGATTGTCTT 401 AA

[0024] The Kozak sequence of protein AOX1 gene of P. pastoris (AAACG) was added to the 5-end of the designed sequence. Finally, the restriction sites for BamHI (GGATCC) and EcoRI (GAATTC) were added to 5 and 3-ends of the designed gene, respectively.

[0025] SEQ ID NO:3 shows the final sequence of the synthetic gene for protein Sm14 production.

TABLE-US-00004 SEQIDNO:3 1 GGATCCAAACGATGTCTTCTTTCTTGGGTAAGTGGAAGTTGTCTGAATCT 51 CACAACTTCGACGCTGTTATGTCTAAGTTGGGTGTTTCTTGGGCTACCAG 101 ACAAATTGGTAACACCGTTACTCCAACCGTTACCTTCACCATGGACGGTG 151 ACAAGATGACTATGTTGACCGAGTCTACCTTCAAGAACTTGTCTGTTACT 201 TTCAAGTTCGGTGAAGAGTTCGACGAAAAGACTTCTGACGGTAGAAACGT 251 TAAGTCTGTTGTTGAAAAGAACTCTGAATCTAAGTTGACTCAAACTCAAG 301 TTGACCCAAAGAACACTACCGTTATCGTTAGAGAAGTTGACGGTGACACT 351 ATGAAGACTACTGTTACCGTTGGTGACGTTACCGCTATCAGAAACTACAA 401 GAGATTGTCTTAAGAATTC

[0026] Following the synthesis of the designed sequence (SEQ ID NO:3), the cloning and later sequencing of the synthetic gene was performed in vector pCR2.1 to confirm the fidelity of the synthesized sequence with the designed sequence.

1.2 Constructions of Plasmids for Protein Sm14 Expression in Pichia pastoris:

[0027] The synthesized gene was cloned in vector pPIC9K where protein Sm14 is expressed without any fusion, allowing its intracellular production.

[0028] Vector pPIC9K (Invitrogen) was chosen for the construction of Sm14 expression plasmid in P. pastoris for the following reasons:

(1) Possibility for use to express intracellular proteins replacing the alpha-factor gene with the gene of interest, through the vector's BamHI restriction site, located before the Kozak sequence and the beginning of translation. For this purpose, it was necessary to recreate the Kozak sequence before the ATG of the ORF to be expressed, according to Sm14's synthetic gene design.
(2) It has the advantage to allow the selection of clones with multiple copies integrated into the genome, by selecting resistance against antibiotic G418. This possibility did not exist with pPIC9, previously used.

[0029] The strategy for building plasmid pPIC9K-Sm14 is described in FIG. 1. According to this strategy, plasmid pCR21-Sm14-MV and vector pPIC9K were digested with restriction enzymes BamHI and EcoRI. After digestion, the DNA fragments were separated by agarose gel electrophoresis and the fragments corresponding to vector pPIC9K and to the synthetic Sm14-MV insert were excised from the agarose gel, purified and bond using T4 DNA ligase. E. coli's DH5 strain was transformed by link reaction and the clones were selected in LB agar medium containing ampicillin. The pDNA of some ampicillin-resistant clones was purified and analyzed by restriction with enzymes BamHI and EcoRI, as well as by DNA sequencing. The construction so obtained was called pPIC9K-Sm14-MV, which expresses protein Sm14 under control of the strong alcohol oxidase 1 (AOX1) promoter, which is induced by methanol (Cregg et al., 1993).

[0030] According to the present invention, another promoter was successfully used in the expression of recombinant proteins in Pichia pastoris, the constituent promoter of glyceraldehyde-3-phosphate dehydrogenase (GAP) gene. For the large scale production of recombinant proteins, the promoter GAP-based expression system is more appropriate than promoter AOX1, because the risks and costs associated with the transport and storage of large volume of methanol are eliminates (Zhang et al, 2009).

[0031] According to the strategy described in FIG. 1, promoter GAP was amplified from the genomic DNA of strain GS115 of Pichia pastoris, using primers GAP-for (SEQ ID NO: 4) and GAP-rev (SEQ ID NO:5), which contain the restriction sites SacI and BamHI, respectively.

TABLE-US-00005 SEQIDNO:4 GAGCTCTTTTTTGTAGAAATGTCTTGG 27 SEQIDNO:5 GGATCCAAATAGTTGTTCAATTGATTGAAATAGG 34

[0032] With these oligonucleotides, a fragment of 506 pairs of bases corresponding to promoter GAP of Pichia pastoris was amplified (SEQ ID NO: 6).

TABLE-US-00006 SEQIDNO:6 GAGCTCTTTTTTGTAGAAATGTCTTGGTGTCCTCGTCCAATCAGGTAGCC 50 ATCTCTGAAATATCTGGCTCCGTTGCAACTCCGAACGACCTGCTGGCAAC 100 GTAAAATTCTCCGGGGTAAAACTTAAATGTGGAGTAATGGAACCAGAAAC 150 GTCTCTTCCCTTCTCTCTCCTTCCACCGCCCGTTACCGTCCCTAGGAAAT 200 TTTACTCTGCTGGAGAGCTTCTTCTACGGCCCCCTTGCAGCAATGCTCTT 250 CCCAGCATTACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCTAGCAG 300 CCCAGGGATGGAAAAGTCCCGGCCGTCGCTGGCAATAATAGCGGGCGGAC 350 GCATGTCATGAGATTATTGGAAACCACCAGAATCGAATATAAAAGGCGAA 400 CACCTTTCCCAATTTTGGTTTCTCCTGACCCAAAGACTTTAAATTTAATT 450 TATTTGTCCCTATTTCAATCAATTGAACAACTATTTTTGAACAACTATTT 500 GGATCC 506

[0033] The amplified promoter GAP's DNA, as well as plasmid pPIC9K-Sm14-MV was digested with restriction enzymes SacI and BamHI. The fragments corresponding to vector pPIC9K-SM14-MV and to insert GAP were purified from agarose gel, purified and bonded. E. coli's DH5 strain was transformed by link reaction and the clones were selected in LB agar medium containing ampicillin. The pDNA of some ampicillin-resistant clones was purified and analyzed by restriction with enzymes BamHI and EcoRI, as well as by DNA sequencing. The construction so obtained was called pGAP9K-Sm14-MV, which expresses the protein under control of constituent promoter pGAP.

1.3 Transformation of P. pastoris with Plasmids pPIC9K-Sm14-MV and pGAP9K-Sm14-MV. and Selection of Recombinant Clones with Multiple Copies

[0034] In order to produce the protein, the GS115 (his4) of P. pastoris strain was transformed with plasmids pPIC9K-Sm14-MV and pGAP9K-Sm14-MV, digested with enzymes SacI. With the DNA digested and purified, the competent cells for electroporation of strain GSI15 were transformed.

[0035] After transformation, the cells were spread in RD medium (histidine-free medium, which contains: 1 M sorbitol; 2% dextrose; 1.34% YNB; 410.sup.5% biotin; and 0.005% of each amino acid: L-glutamate, L-methionine, L-lysine, L-leucine and L-isoleucine for the selection of strains transformed by auxotrophy marker his4. Clones that managed to grow in the histidine-free medium were submitted to selection with antibiotic G418, at the concentrations of 0.5; 1; 2; and 4 mg/ml, in a YPD culture (1% Yeast Extract, 2% Peptone; 2% dextrose) at 30 C.

[0036] In order to confirm whether the selected clones presented the expression cassette of the synthetic gene, genomic DNA of the clones was purified and used in PCR reactions with primers AOX5 and AOX3 (for pPIC9K-Sm14-MV) and GAPS' and AOX3 (for pGAP9K-Sm14-MV).

1.4 Cultivation Expression in Shaker

[0037] In order to test the expression of protein Sm14 in P. pastoris/pPIC9K-Sm14-MV clones, the clones were grew in shaker in 5 mL of the BMG medium (Buffered Minimal Glycerol medium, containing: 1.34% YNB; 0.04% biotin, 0.1 M potassium phosphate pH 6.0; and 1% glycerol) at 30 C. for 48 hours and then transferred to a BMM medium, 5 ml, (Buffered Minimal Methanol medium, containing the same components as the BMG medium, except for glycerol which was replaced with 0.5% methanol), for the induction of recombinant protein expression. After 72 hours, adding 0.5% methanol every 24 hours, the total proteins of each clone were analyzed by SDS-PAGE (FIG. 2). FIG. 2 shows the result of inducing the expression of protein Sm14 in P. pastoris clones GS115/pPIC9K-Sm14-MV.

M.Low Molecular Weight Marker

[0038] Sm14.Control of protein Sm14 without purified fusion of E. coli
1 to 8.Total protein of clones 1-17 GS115/pPIC9K-Sm14-MV after induction with methanol.

[0039] In order to test the expression of protein Sm14 in P. pastoris/pGAP9K-Sm14-MV clones, the clones were grown in 5 ml of the BMG medium for only 72 hours. FIG. 3 shows the result of protein Sm14 expression in P. pastoris GS115/pGAP9K-Sm14-MV clones.

M.Low Molecular Weight Marker

[0040] 1 to 6.Total protein of clones 1-17 GSI15/pGAP9KSm14-MV following 3 days of culture in BMG medium.
Sm14.Control of protein Sm14 without purified fusion of E. coli

[0041] In all the selected clones, it was possible to note a majority band which coincides with the size of purified E. coli purified fusionless Sm14 protein.

1.5 Expression of Sm14 Recombinant in P. pastoris GS115/pPIC9K-Sm14-MV in Fermenter.

[0042] Fermentation was conducted with a 5-Liter culture in a fermenter with automatic supply, pH, antifoaming adjustments. The fermentation medium used was Basal Salt Medium (BSM) supplemented with the metal salt solutions (trace elements) and biotin.

[0043] In order to prepare the inoculum, an ampoule from the working bank of Pichia pastoris GS115/pPIC9K-Sm14-MV strain was used to inoculate the YDP medium and cultivated in shaker for 18 hours at 220 rpm, 30 C. Subsequently, the cells are inoculated in BSM medium supplemented with glycerol, cultivate in shaker for 18 hours before inoculating the fermenter.

[0044] The first fermentation phase (Glycerol fed batch) is started by adding 50% glycerol containing trace elements (12%) to the culture. The temperature and pH were maintained at 30 C. and 5.00, respectively and aeration in 1.0 VVM.

[0045] Induction was starting by adding 100% methanol, containing trace elements (12%) to the fermenter. Following the adaptation period, methanol was added according to the dissolved oxygen behavior.

[0046] FIG. 5 shows the induction of protein Sm14 expression in P. pastoris GS115/pPIC9K-Sm14-MV, in fermenter, where:

M.Low Molecular Weight Marker

[0047] 1-7.Induction of expression for 24, 48, 72 and 96, 120, 144 and 168 hours, respectively.
Sm14.Control of protein Sm14 without purified fusion of E. coli

[0048] Thus, the recombinant protein-specific induction corresponding to Sm14 in P. pastoris in the cultivation in fermentation was proven.

[0049] The yield of wet molecular mass reached of 170 g per liter and the estimated yield of Sm14 is of approximately 1 g of protein per culture liter. By changing the fermentation conditions, it is possible to exceed the cell mass yield and, correspondingly the yield obtained of protein Sm14.

[0050] Even so, the obtained value already corresponds to a high protein Sm14 expression level, which comprises the majority protein of the cells following induction with methanol.

2. Purification of Recombinant Protein Sm14 Expressed in P. pastoris GSI15/pPIC9K-Sm14-MV STRAIN

[0051] The purification protocol for recombinant protein Sm14 from P. pastoris cytoplasm was based on the methodology developed at the Experimental Schistosomiasis Laboratory of the Oswaldo Cruz Institute (IOC) for Sm14 purification without fusion into the E. coli system.

[0052] Lysis:

[0053] The purification of recombinant protein starts with the lysis of P. pastoris cells. For that purpose, the cells are resuspended 30 mM Tris-HCl 30 mM pH 9.5 and lysed through pressure at 1500 Bar. The lysate is clarified by centrifugation.

[0054] Conditioning:

[0055] The clarified lysate is submitted to tangential filtration in the 100 kDa membrane in order to remove high molecular weight proteins, followed by concentration and diafiltration in the 3 kDa membrane with buffer 30 mM Tris-HCl 30 mM pH 9.5 until conductivity corresponds to the buffer value.

[0056] Capture:

[0057] Clarified lysate is loaded in resin Q-Sepharose XL (GE Healthcare) resin, balanced with buffer A (30 mM Tris-HCl pH 9.5). Then, the culture is washed with buffer A, and the protein is eluted with buffer B (30 mM Tris-HCl pH 8.0) where protein Sm14 elutes with a higher purity degree.

[0058] Polishing:

[0059] The eluted protein of resin Q-sepharose XL presents few contaminant proteins. In order to separate such proteins from protein Sm14, the gel-filtration in resin Sephacryl S100 HR (GE Healthcare) was used, using PBS pH 7.4 as mobile phase.

4. Analysis of Recombinant Protein Sm14 Produced in Pichia pastoris

[0060] The recombinant protein was purified from P. pastoris by using the same physical-chemical characteristics as protein Sm14-MV without fusion expressed in E. coli. The protein so purified from P. pastoris corresponds in size with the protein specifically induced by methanol. Since we have the synthetic gene of protein Sm14-MV under control of AOX1 promoter in the expression cassette, we deduce that the expressed and purified P. pastoris protein is protein Sm14-MV.

[0061] In order to confirm this statement, P. pastoris' purified protein was analyzed by western blot, by using rabbit anti-Sm14 serum (FIG. 6). FIG. 6 represents the western blot analysis of P. pastoris' purified protein.

1 and 2Protein Sm14-MV purified from P. pastoris
3.Positive control of protein Sm14 without purified fusion of E. co/i

[0062] In this experiment, it was possible to observe that anti-Sm14 antibodies specifically recognized P. pastoris' purified protein (rabbit serum does not recognize endogenous P. pastoris proteins, data not shown), therefore confirming its identity.

[0063] Finally, it was necessary to identify whether the P. pastoris purified protein has a structure which corresponds to beta folding, characteristic of proteins of the Fatty Acid Binding Protein family, to which Sm14 belongs. To do so, protein samples were analyzed by circular dichroism, using spectral photopolarimeter J-815 (JASCO) (FIG. 7). FIG. 9 shows the circular dichroism spectrum of P. pastoris' purified protein Sm14.

[0064] As it can be noted in FIG. 7, the spectrum corresponds to a beta-structure protein. This spectrum was similar to circular dichroism spectra of lots of protein Sm14 previously purified from E. coli in laboratory.

[0065] Thus, based on the report above we may conclude that this invention allows us to:

[0066] design and synthesize a synthetic gene for high expression of protein Sm14-MV in Pichia pastoris;

[0067] build pPIC9K-Sm14-MV and pGAP9K-Sm14-MV expression plasmids containing the synthetic gene's sequence under control of AOX1 and GAP promoters, respectively;

[0068] obtain P. pastoris strains producing protein Sm14; and,

[0069] purify protein Sm14 in two chromatographic steps, feasible for scheduling for industrial production.

[0070] Through the process of the present invention, it was possible to build the pPIC9K-Sm14-MV and pGAP9K-Sm14MV plasmids for the constituent expression of protein Sm14 in Pichia pastoris and select the producing clones from the transformation of strain GS115 with plasmid pGAP9K-Sm14MV. One of the advantages of the invention is that the strains produce the protein within a lower time compared to the methanol induction system in constituent way. In addition, the new strain simplifies the fermentation process for the production of protein Sm14.

[0071] Therefore, the present invention described herein shows that protein Sm14 provides protection against Schistosoma mansoni infection in mice, in E. coli and P. pastoris platforms.

REFERENCES

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