Patent Application: US-201113824378-A

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:
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 . 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 ). 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 . 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 . 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 . 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 . 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 fig1 . 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 fig2 . in fig2 we show the results of sm14 - mv cloning in ppic9k ; clones were selected as shown below : in fig2 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 . fig3 shows the pcr analysis of p . pastoris gs115 clones transformed with ppic9k - sm14 - mv and selected with 4 mg / ml of g418 . as shown in fig3 , 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 ( fig4 ). fig4 shows the results of inducing the expression of sm14 in p . pastoris clones gs115 / ppic9k - sm14 - mv . 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 fig5 shows the induction of sm14 expression in p . pastoris gs115 / ppic9k - sm14 - mv , where : 2 to 7 .— induction of expression for 0 , 24 , 48 , 72 and 91 hours , respectively , in bmm medium 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 ( fig5 , 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 ( fig5 , lanes 2 to 7 ). methanol was added to the culture every 24 hours , to achieve a final concentration of 0 . 5 %. as fig5 shows , in the bmg medium we did not obtain protein expression induced with methanol ( fig5 , lane 1 ), and neither in time zero with bmm inducing medium ( fig5 , 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 ( fig6 , 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 ( fig6 , 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 ) ( fig6 , 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 ( fig6 , lane 8 ) presented the same retention volume as fusionless proteins produced in e . coli ( fig7 ). fig7 shows the results of the gel - filtration chromatogram of the sm14 - mv protein produced in p . 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 ( fig8 ). fig8 represents the western blot analysis of p . pastoris &# 39 ; purified 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 ) ( fig9 ). fig9 shows the circular dichroism spectrum of p . pastoris &# 39 ; purified sm14 protein . as one can see in fig9 , 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 ; 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 . 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