Abstract:
The production of recombinant proteins using a synthetic gene for increased expression of the protein in  Pichia pastoris  is disclosed. More specifically, disclosed is a process for producing a recombinant Sm14 protein of  Schistosoma mansoni , by providing a synthetic gene for increased production of Sm14 protein, producing the gene and genetically manipulating the  Pichia pastoris  strain with the gene in order to produce a vaccine effectively. Improved methods are also provided for producing and purifying this protein from  P. pastoris  cells, which can be scaled for industrial production.

Description:
SEQUENCE LISTING STATEMENT 
     Incorporated herein by reference in its entirety is a Sequence Listing named “B120420017_SeqLst_ST25.txt”, which was submitted to the USPTO via EFS-web on Mar. 17, 2013 as an ASCII text file 9 KB in size. This file, which was created Mar. 17, 2013, constitutes both the paper and computer readable form of the Sequence Listing. 
     FIELD OF APPLICATION 
     The present invention is related to the field of recombinant protein production using a synthetic gene associated with high protein expression in  Pichia pastoris . More specifically, this invention describes the production of Sm14  Schistosoma mansoni  recombinant protein. A synthetic gene was created to promote high Sm14 expression, and with this gene we obtained and genetically manipulated a  Pichia pastoris  strain for effectively producing a vaccine. We have also improved that protein&#39;s production and purification processes from  P. pastoris  cells; the industrial production of such processes may be scheduled. 
     INVENTION FUNDAMENTALS 
     The Sm14 protein&#39;s molecular weight is approximately 14.8 kDa and it is significantly similar to proteins that belong to the protein family which binds to fatty acids. It has been widely studied and described by applicant in his previous patent applications. 
     The three-dimensional structure of protein Sm14 was predicted through molecular modeling by computerized homology, as well as crystallography and Nuclear Magnetic Resonance. The structure of protein Sm14 allowed us to identify potential protective epitopes and enabled us to use rSm14 as a vaccination antigen. 
     Sm14&#39;s structure, as well as models built for homologous  Fasciola hepatica  proteins (FABP type 3 is the one with greater shared sequential identity, 49%), shows that those molecules adopt three-dimensional configurations that are similar to molecules of other protein families that bind to lipids (Fatty Acid Binding Proteins—FABP). 
     Therefore, based on the entire state of the art knowledge gathered by inventors, we will demonstrate here how Sm14 recombinant forms can provide significant protection against infections caused by supposedly pathogenic heltminths in relation to humans and animals. 
     In papers which demonstrated Sm14&#39;s protective activity for the first time, the corresponding recombinant protein was expressed with the pGEMEX-Sm14 vector as inclusion bodies. After bodies were isolated and washed, the protein was purified by preparative electrophoresis of the corresponding band (Tendler et al., 1996) by electroelution. However, this methodology was not adequate for producing proteins in a larger scale. Later, Sm14 started being produced with a fusion of six consecutive histidines (6×His) in the extreme amino-terminus of the  Escherichia coli  expression system, as inclusion bodies. After bodies were obtained and solubilized, refold was necessary in order to obtain a functional and immunologically active protein (Ramos et al., 2001). 
     Therefore, despite all the knowledge gathered by inventors, there are still disadvantages to be overcome in order to obtain antigenic material that may be obtained with high performance, at an industrial scale and under GMP conditions, and that will not lose its stability feature. 
     INVENTION SUMMARY 
     This invention proposes a platform for producing a recombinant vaccine against heltminths in  Pichia pastoris . Through the abovementioned platform it is possible to obtain a recombinant vaccine against heltminths (in  P. pastoris ), including the production and purification processes of Sm14 developed in the  Pichia pastoris  system. 
     The invention also proposes a synthetic gene for Sm14 protein expression.  Pichia pastoris &#39; genetic transformation with this synthetic gene under control of the AOX1 promoter allows one to produce and purify Sm14. 
     Therefore, the invention allows us to obtain Sm14 from a synthetic gene containing codons optimized for high expression in  Pichia pastoris , SEQ ID NO:3 (final gene sequence), as well as Sm14 purification procedures. 
    
    
     
       A BRIEF DESCRIPTION OF FIGURES 
         FIG. 1  shows the strategy for building the pPIC9K-Sm14-MV plasmid. 
         FIG. 2  shows the cloning of Sm14-MV into pPIC9K. 
         FIG. 3  shows the PCR analysis of  P. pastoris  clones. 
         FIG. 4  shows the induction of Sm14 expression in  P. pastoris  clones GS115/pPIC9K-Sm14-MV. 
         FIG. 5  shows the induction of Sm14 expression in  P. pastoris  GS115/pPIC9K-Sm14-MV. 
         FIG. 6  shows the results of Sm14 purification. 
         FIG. 7  shows the results of the gel-filtration chromatogram of the Sm14-MV protein produced in  P. pastoris.    
         FIG. 8  shows a western blot analysis of the  P. pastoris  purified protein. 
         FIG. 9  shows the circular dichroism spectrum of the  P. pastoris  purified Sm14 protein. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The main purpose of this invention is to produce a recombinant vaccine against heltminths. This goal 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 Sm14 expression, and with this gene we obtained a  Pichia pastoris  strain for effectively producing a vaccine. This invention also includes the protein&#39;s production and purification processes from  P. pastoris  cells; such processes may be scheduled for industrial production. 
     The expression system in methylotrophic yeast  Pichia pastoris  has significant advantages when compared with systems based on  E. coli  for producing recombinant proteins at an industrial scale. Among such advantages we can mention, for instance, the stability of transformed strains, high expression, high cell-density culture, easy culture scheduling, no human health hazards, and it does not produce endotoxins (Fabe et al., 1995). The latter advantage is one of the factors which influenced the change of micro-organism for protein expression. This is because the need for detecting or quantifying endotoxins produced by Gram-negative bacteria in each batch would be a limiting factor, since products generated in  E. coli  that will later be used in human beings must be free from bacterial endotoxins. This additional requirement would make  E. coli  production processes difficult, and this would have a negative impact on final production costs. 
     The invention will now be described through its best execution process. 
     1. Obtaining the  Pichia pastoris  Recombinant Strain 
     1.1 Synthetic Gene for Sm14 Expression in  P. pastoris:    
     First a gene was designed and synthesized containing codons that were optimized to obtain maximum Sm14 expression in  P. pastoris . In our case we used Sm14-MV; however any form of Sm14 can be used. 
     There is evidence in literature about differential use of codons between proteins with low and high expression levels in the same organism (Roymondal and Sahoo, 2009). However, codon usage tables available in databases (for example: www.kazusa.or.jp/codon) contain data from all body proteins, and do not take the level of gene expression into account. For this reason, in order to design the gene we initially drew up a codon usage table based on data about sequences that codify recombinant proteins expressed above 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 1 
               
             
             
               
                   
               
               
                 List of high-expression recombinant proteins in  P. pastoris . 
               
             
          
           
               
                 Expressed 
                   
                   
               
               
                 Protein 
                 (mg/L) 
                 Reference 
               
               
                   
               
             
          
           
               
                 Hydroxynitrile 
                 22000 
                 Hasslacher, M. et al. (1997) 
               
               
                 lyase 
                   
                 Protein 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) 
               
               
                 fragment C 
                   
                 Bio/Technology 9: 455-460 
               
               
                 Human tumor 
                 10000 
                 Sreekrishna, K. et al. (1989) 
               
               
                 necrosis factor 
                   
                 Biochemistry 28: 4117-4125 
               
               
                 α-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. 
               
               
                   
               
             
          
         
       
     
     For gene design we chose the sequence of Sm14-MV protein, which has a valine residue at position 62-replacing cystein, which makes it more stable (Ramos et al., 2009); it is represented here as SEQ ID NO:1. 
     
       
         
               
               
             
           
               
                   
                 SEQ ID NO: 1 
               
               
                   
                 MSSFLGKWKL SESHNFDAVM SKLGVSWATR QIGNTVTPTV 
               
               
                   
                   
               
               
                   
                 TFTMDGDKMT MLTESTFKNL SVTFKFGEEF DEKTSDGRNV 
               
               
                   
                   
               
               
                   
                 KSVVEKNSES KLTQTQVDPK NTTVIVREVD GDTMKTTVTV 
               
               
                   
                   
               
               
                   
                 GDVTAIRNYK RLS 
               
             
          
         
       
     
     After the first selection of codons according to the table drawn up with protein data from Table 1, we performed sequence depurations which resulted in donor transcription termination sites (ATTTA) and splicing cryptic receptors (MAGGTRAGT and YYYNTAGC, respectively) and repetitive sequences (cleavage sites for restriction enzymes BamHI and EcoRI). SEQ ID NO:2 shows the sequence designed to express Sm14-MV protein in  Pichia pastoris . 
     
       
         
               
               
             
               
               
               
             
           
               
                 SEQ ID NO: 2 
                   
               
             
          
           
               
                 1 
                 ATGTCTTCTT TCTTGGGTAA GTGGAAGTTG TCTGAATCTC ACAACTTCGA 
                   
               
               
                   
               
               
                 51 
                 CGCTGTTATG TCTAAGTTGG GTGTTTCTTG GGCTACCAGA CAAATTGGTA 
               
               
                   
               
               
                 101 
                 ACACCGTTAC TCCAACCGTT ACCTTCACCA TGGACGGTGA CAAGATGACT 
               
               
                   
               
               
                 151 
                 ATGTTGACCG AGTCTACCTT CAAGAACTTG TCTGTTACTT TCAAGTTCGG 
               
               
                   
               
               
                 201 
                 TGAAGAGTTC GACGAAAAGA CTTCTGACGG TAGAAACGTT AAGTCTGTTG 
               
               
                   
               
               
                 251 
                 TTGAAAAGAA CTCTGAATCT AAGTTGACTC AAACTCAAGT TGACCCAAAG 
               
               
                   
               
               
                 301 
                 AACACTACCG TTATCGTTAG AGAAGTTGAC GGTGACACTA TGAAGACTAC 
               
               
                   
               
               
                 351 
                 TGTTACCGTT GGTGACGTTA CCGCTATCAG AAACTACAAG AGATTGTCTT 
               
               
                   
               
               
                 401 
                 AA 
               
             
          
         
       
     
     We added the Kozak sequence of the AOX1 protein gene of  P. pastoris  (AAACG) to the 5″-end of the designed sequence. Finally, we added restriction sites for BamHI (GGATCC) and EcoRI (GAATTC) to 5′ and 3′-ends of the designed gene, respectively. 
     SEQ ID NO:3 shows the final sequence of the synthetic gene for Sm14 protein production. 
     
       
         
               
               
             
               
               
               
             
           
               
                 SEQ ID NO: 3 
                   
               
             
          
           
               
                 1 
                 GGATCCAAAC GATGTCTTCT TTCTTGGGTA AGTGGAAGTT GTCTGAATCT 
                   
               
               
                   
               
               
                 51 
                 CACAACTTCG ACGCTGTTAT GTCTAAGTTG GGTGTTTCTT GGGCTACCAG 
               
               
                   
               
               
                 101 
                 ACAAATTGGT AACACCGTTA CTCCAACCGT TACCTTCACC ATGGACGGTG 
               
               
                   
               
               
                 151 
                 ACAAGATGAC TATGTTGACC GAGTCTACCT TCAAGAACTT GTCTGTTACT 
               
               
                   
               
               
                 201 
                 TTCAAGTTCG GTGAAGAGTT CGACGAAAAG ACTTCTGACG GTAGAAACGT 
               
               
                   
               
               
                 251 
                 TAAGTCTGTT GTTGAAAAGA ACTCTGAATC TAAGTTGACT CAAACTCAAG 
               
               
                   
               
               
                 301 
                 TTGACCCAAA GAACACTACC GTTATCGTTA GAGAAGTTGA CGGTGACACT 
               
               
                   
               
               
                 351 
                 ATGAAGACTA CTGTTACCGT TGGTGACGTT ACCGCTATCA GAAACTACAA 
               
               
                   
               
               
                 401 
                 GAGATTGTCT TAAGAATTC 
               
             
          
         
       
     
     After synthesizing the designed sequence (SEQ ID NO:3), we performed cloning and later sequencing of the synthetic gene in vector pCR2.1 to confirm the synthesized sequence was faithful to the designed sequence. 
     1.2 Plasmid Construction for Sm14 Expression in  Pichia pastoris:    
     The synthesized gene was cloned in vector pPIC9K where protein Sm14 is expressed without any fusion, making its intracellular production possible. 
     Vector pPIC9K (Invitrogen) was chosen for construction of the Sm14 expression plasmid in  P. pastoris  for the following reasons: 
     (1) It may be used to express intracellular proteins replacing the alpha-factor gene with the gene of choice, through the vector&#39;s BamHI restriction site, located before the Kozak sequence and the beginning of translation. In order to do this it was necessary to recreate the Kozak sequence before the ATG of the ORF to be expressed, according to the design of Sm14&#39;s synthetic gene. 
     (2) It offers the advantage of allowing a selection of clones with multiple copies integrated into the genome, by selecting resistance to antibiotic G418. There was no such possibility with pPIC9, used previously. 
     The strategy for building the pPIC9K-Sm14 plasmid is described in  FIG. 1 . 
     With plasmid pCR21-Sm14-MV, we changed the DH5 α E. coli  strain for its propagation. Afterward pDNA was purified with Qiaprep Spin Miniprep Kit (QIAGEN). This plasmid, as well as vector pPIC9K, was digested simultaneously with restriction enzymes BamHI and EcoRI (both of New England Biolabs). After digestion, DNA fragments were separated by agarose gel electrophoresis containing ethidium bromide. Fragments corresponding to vector pPIC9K and to the synthetic Sm14-MV insert were excised from the agarose gel and purified with a QIAquick Gel Extraction Kit (QIAGEN). 
     Purified fragments were linked using T4 DNA ligase (New England Biolabs).  E. coli &#39;s DH5α strain was transformed by the link reaction and clones were selected in LB agar medium containing ampicillin. The pDNA of a few ampicillin-resistant clones was purified and analyzed by restriction with enzymes BamHI and EcoRI, shown in  FIG. 2 . In  FIG. 2  we show the results of Sm14-MV cloning in pPIC9K; clones were selected as shown below: 
     M.—1 Kb DNA ladder+phiX174/HaeIII 
     1.—pPIC9K/BamHI+EcoRI 
     2.—pCR21—Sm14-MV/BamHI+EcoRI 
     3 to 11.—Clones pPIC9K-Sm14-MV/BamHI+EcoRI. 
     In  FIG. 2  arrows mark the insert position and cloning vector. 
     Clones that showed bands corresponding to inserts were selected and sequenced with AOX5′ primer to confirm successful cloning and the sequence&#39;s fidelity. Therefore, the synthetic sequence for Sm14 expression remained under control of strong alcohol oxidase 1 promoter (AOX1), which is induced by methanol (Cregg et al., 1993). 
     1.3 Transformation of  P. pastoris  with Plasmid pPIC9K-Sm14-MV and Selection of Recombinant Clones with Multiple Copies 
     In order to produce the protein, the GS115 (his4)  P. pastoris  strain was transformed with plasmid pPIC9K-Sm14-MV. The latter was purified with the Maxiprep(Qiagen) kit. Plasmid DNA was digested separately with enzymes BglII and Sad (New England Biolabs), using 20 μg of DNA for each reaction. Digestion reactions were separated by agarose gel electrophoresis and bands with DNA fragments containing Sm14-MV&#39;s synthetic gene were severed from the gel and DNA was purified. 
     Electroporation-competent cells for the GS115 strain were prepared and transformed separately with purified DNA from restriction reactions for BglII and Sad (10 μg of DNA per transformation). Digestion with enzyme BglII guides the recombination of the expression cassette of AOX1&#39;s gene, in  P. pastoris  genome, while digestion with Sad can be integrated in other regions. 
     After transformation, cells were spread in to RD medium (histidine free medium, which contains: 1 M sorbitol; 2% dextrose; 1.34% YNB; 4×10 −5 % biotine; and 0.005% of each amino acid: L-glutamate, L-methionine, L-lysine, L-leucine and L-isoleucine for 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 concentrations: 0.5; 1; 2; and 4 mg/ml, in a YPD culture (1% Yeast Extract, 2% Peptone; 2% dextrose) at 30° C. in microculture plates. Only clones transformed with plasmid pPIC9K-Sm14-MV digested with enzyme Sad managed to grow with G418 at 4 mg/ml; this characterizes the insertion of multiple copies of the expression cassette into  P. pistoris &#39; genome. 
     In order to confirm whether selected clones had the expression cassette of the synthetic gene, genomic DNA of 17 clones was purified and used in PCR reactions with primers AOX5 and AOX3′. Plasmid pPIC9K-Sm14-MV was used as positive control.  FIG. 3  shows the PCR analysis of  P. pastoris  GS115 clones transformed with pPIC9K-Sm14-MV and selected with 4 mg/ml of G418. 
     M.—phiX174/HaeIII 
     1 to 17.—Clones GS115/pPIC9K-Sm14-MV 
     18.—Positive control: plasmid pPIC9K-Sm14-MV. 
     As shown in  FIG. 3 , all clones selected with 4 mg/ml of G418 presented the sequence synthetic Sm14-MV. 
     In order to test the expression of Sm14protein, clones grew in a BMG medium (Buffered Minimal Glycerol medium, containing: 1.34% YNB; 0.04% biotine, 0.1 M potassium phosphate pH 6.0; and 1% glycerol) for 48 hours and then were transferred to a BMM medium (Buffered Minimal Methanol medium, containing the same components as the BMG medium, except for glycerol which was replaced with 0.5% methanol and additional EDTA for the final concentration of 1 mM), for induction of expression of the recombinant protein. After 72 hours, adding 0.5% methanol every 24 hours, total proteins of each clone were analyzed by SDS-PAGE ( FIG. 4 ).  FIG. 4  shows the results of inducing the expression of Sm14 in  P. pastoris  clones GS115/pPIC9K-Sm14-MV. 
     M.—Low Molecular Weight Marker 
     1.—Positive control Sm14 purified fusionless  E. coli  protein 
     2 to 18—Total protein of clones 1-17 GS115/pPIC9K-Sm14-MV after induction with methanol. 
     1.4 Induction of Recombinant Sm14 Expression in  P. pastoris  GS115/pPIC9K-Sm14-MV 
       FIG. 5  shows the induction of Sm14 expression in  P. pastoris  GS115/pPIC9K-Sm14-MV, where: 
     M.—Low Molecular Weight Marker 
     1.—Non-induced sample, in BMG medium 
     2 to 7.—Induction of expression for 0, 24, 48, 72 and 91 hours, respectively, in BMM medium 
     8.—Positive control Sm14 purified fusionless  E. coli  protein 
     It was possible to observe a majority band in all selected clones which coincides with the size of purified  E. coli  purified fusionless Sm14 protein. 
     In order to confirm whether the protein was induced by methanol, clone #1 grew in a BMG medium for 48 hours ( FIG. 5 , lane 1) at 250 rpm, at 30° C.; it was later transferred to a BMM medium (0.5% methanol). We collected samples at different time intervals ( FIG. 5 , lanes 2 to 7). Methanol was added to the culture every 24 hours, to achieve a final concentration of 0.5%. 
     As  FIG. 5  shows, in the BMG medium we did not obtain protein expression induced with methanol ( FIG. 5 , lane 1), and neither in time zero with BMM inducing medium ( FIG. 5 , lane 2). Induction was visible after 24 hours of culture in BMM medium and it remained stable during cell growth, until 91 hours which was the timeframe of the experiment. 
     Therefore, we have verified the specific induction of recombinant protein corresponding to Sm14 in  P. pastoris.    
     In our experiment, by using only BMM medium and methanol at 0.5%, it was possible to achieve 26 grams of wet cell mass per liter of culture, maintaining a good level of expression of protein Sm14, which is cells&#39; major protein after methanol induction. In addition to using a fermenter and more adequate means for producing recombinant proteins in  P. pastoris , it is possible to obtain both greater cell mass and higher Sm14 expression. 
     2. Purification of SM14 Recombinant Protein Expressed in  P. pastoris  Strain GS115/pPIC9K-Sm14-MV 
     The purification protocol for recombinant Sm14 from  P. pastoris  cytoplasm was based on methodology developed at the Experimental Schistosomiasis Laboratory of the Oswaldo Cruz Institute (IOC) for Sm14 purification without fusion into the  E. coli  system. 
     Lysis: Purification of recombinant proteins begins with lysis of  P. pastoris  cells. To that end, cells are resuspended 30 mM Tris-HCl 30 mM pH 9.5 and French press lysed. Lysate is clarified by centrifugation ( FIG. 6 , lane 2). 
     Capture: Clarified lysate is loaded in resin Q-Sepharose XL (GE Healthcare), balanced with buffer A (30 mM Tris-HCl pH 9.5). All Sm14 protein of lysate is absorbed by the resin, since there is no Sm14 protein in the material that is not absorbed in the resin ( FIG. 6 , lane3). After the protein is loaded, the column is washed with buffer A. Protein is eluted with buffer B (30 mM Tris-HCl pH 8.0) in the AKTA-fplc system (GE Healthcare) ( FIG. 6 , lanes 4-6). 
     Polishing: Eluted protein of resin Q-sepharose XL presents few contaminant proteins. In order to separate those proteins from Sm14 we used gel-filtration. To do so, fractions of the ion exchange chromatography containing Sm14 were gathered and concentrated in the Centriprep YM-10 membrane (Millipore) and the material was applied to column Sephacryl 5100 HR 26/60 (GE Healthcare) using PBS pH 7.4 as the mobile phase. The chromatographic peak ( FIG. 6 , lane 8) presented the same retention volume as fusionless proteins produced in  E. coli  ( FIG. 7 ). 
       FIG. 6  shows the results of Sm14 purification, where 
     M.—Low Molecular Weight Marker 
     1.—Positive control Sm14 purified fusionless  E. coli  protein 
     2.—Clarified lysate, 2.—Proteins not absorbed in resin Q-sepharose XL 
     3-6.—Resin Q-sepharose XL eluted fractions 
     7.—Pool of ion exchange chromatographic fractions 
     8.—Gel-filtration peak in column sephacryl S-100 HR 26/60 
       FIG. 7  shows the results of the gel-filtration chromatogram of the Sm14-MV protein produced in  P. pastoris.    
     4—Analysis of Recombinant Sm14 Produced in  Pichia pastoris    
       P. pastoris  recombinant protein was purified using the same physical-chemical characteristics as the fusionless Sm14-MV protein expressed in  E. coli . The purified protein of this form of  P. pastoris  corresponds in size to the protein specifically induced by methanol. Since we have the synthetic gene of Sm14-MV under control of AOX1 promoter in the expression cassette, we deduce that the expressed and purified  P. pastoris  protein is Sm14-MV. 
     In order to confirm this statement,  P. pastoris &#39; purified protein was analyzed by western blot, using rabbit anti-Sm14 serum ( FIG. 8 ).  FIG. 8  represents the western blot analysis of  P. pastoris &#39; purified protein. 
     1 and 2 .—P. pastoris &#39; purified Sm14-MV protein 
     3.—Positive control Sm14 purified fusionless  E. coli  protein 
     In this experiment we could observe that anti-Sm14 antibodies specifically recognized  P. pastoris &#39; purified protein (rabbit serum does not recognize endogenous  P. pastoris  proteins, data not shown), therefore confirming its identity. 
     Finally, it was necessary to identify whether the  P. pastoris  purified protein has a structure which corresponds to beta folding, which is typical 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. 9 ). 
       FIG. 9  shows the circular dichroism spectrum of  P. pastoris &#39; purified Sm14 protein. 
     As one can see in  FIG. 9 , the spectrum corresponds to a beta-structure protein. This spectrum was similar to circular dichroism spectra of lots of  E. coli  Sm14 protein previously purified in the Experimental Schistosomiasis Laboratory. 
     Thus, based on the report above we may conclude that this invention will allow us to: 
     design and synthesize a synthetic gene for high expression of Sm14-MV in  Pichia pastoris;    
     build a pPIC9K-Sm14-MV expression plasmid that contains the synthetic gene&#39;s sequence under control of AOX1 promoter; 
     obtain a  P. pastoris  strain that produces Sm14 and; 
     purify Sm14 in two chromatographic stages, whose scheduling for industrial production is feasible. 
     Therefore, the invention described herein shows that the Sm14 protein protects against infections caused by  Schistosoma mansoni  in mice, in platforms  E. coli  and  P. pastoris.    
     REFERENCES 
     
         
         (1) Cregg, J. M., Vedvick, T. S, and Raschke, W. C. Recent advances in the expression of foreign genes in  Pichia pastoris . BioTechnology v. 11, p. 905-910, 1993. 
         (2) Faber, K. N., Harder, W., and Veenhuis, M. Review: Methylotropic Yeasts as Factories for the Production of Foreign Proteins. Yeast. v. 11, p. 1331-1344, 1995. 
         (3) Ramos, C. R., Spisni, A., Oyama, S. Jr., Sforca, M. L., Ramos, H. R, Vilar, M. M., Alves, A. C., Figueredo, R. C., Tendler, M., Zanchin, N. I., Pertinhez, T. A., Ho, P. L. Stability improvement of the fatty acid binding protein Sm14 from  S. mansoni  by Cys replacement: structural and functional characterization of a vaccine candidate.  Biochim Biophys Acta. v.  1794, p. 655-662, 2009. 
         (4) Ramos, C. R., Vilar, M. M., Nascimento, A. L., Ho, P. L., Thaumaturgo, N., Edelenyi, R., Almeida, M., Dias, W. O., Diogo, C. M., Tendler, M. r-Sm14-pRSETA efficacy in experimental animals. Mem Inst Oswaldo Cruz.v. 96, p. 131-135, 2001. 
         (5) Roymondal, U. D. and Sahoo, S. S. Predicting gene expression level from relative codon usage bias: an application to  Escherichia coli  genome. DNA Res. v. 16, p. 13-30, 2009. 
         (6) Tendler, M., Brito, C. A., Vilar, M. M., Serra-Freire, N., Diogo, C. M., Almeida, M. S., Delbem, A. C., Da Silva, J. F., Savino, W., Garratt, R. C., Katz, N., Simpson, A. S. A  Schistosoma mansoni  fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine. Proc Natl Acad Sci USA. v. 93, p. 269-273, 1996.