Patent Application: US-61561503-A

Abstract:
a method of efficiently expressing plasmodium ama - 1 ectodomain or a functional part , derivative , and / or analogue thereof , in a eukaryotic expression system . preferably , the plasmodium ama - 1 ectodomain is pf ama - 1 ectodomain . this protein may be expressed in yeast , such as pichia pastoris . efficient expression is possible using a method for producing mrna encoding the plasmodium ama - 1 ectodomain in a yeast cell , comprising providing the yeast cell with a nucleic acid encoding plasmodium ama - 1 ectodomain , the nucleic acid being modified to utilize the yeast cell &# 39 ; s codon usage . preferably , at least one putative yeast polyadenylation consensus sequence in the nucleic acid has been modified . more preferably , also at least one site in the protein that is generally glycosylated by eukaryotic expression systems , has been removed .

Description:
the following examples illustrate the present invention . the examples do not limit the present invention in any way . a person skilled in the art can perform alternative ways which are still within the scope of the present invention . development of synthetic gene for p . falciparum fvo strain pf ama - 1 cryopreserved parasite stocks from p . falciparum fvo were prepared from an infected aotus lemurinus griseimembra monkey at the young ring stage of development and dna was isolated ( gentra systems inc ., minneapolis , minn .) directly from a parasite stock according to the manufacturer &# 39 ; s instructions . pf ama - 1 was amplified by polymerase chain reaction using pfu polymerase ( promega , leiden , the netherlands ) and primers pf83a : 5 ′- gggggatccatgagaaaattatactgcgtatt - 3 ′ ( nt 1 - 23 and additional bamhi restriction site , seq id no : 1 ) and pf83b : 5 ′- acgtggatccttaatagtatggtttttccatcagaactgg - 3 ′ ( complementary to nt 1843 - 1869 and additional bamhi restriction site , seq id no : 2 ) containing bamhi restriction sites to facilitate cloning in pbluescript . a pool of four independent clones was used for sequence analysis using an abi prism ™ 310 automated sequencer ( pe applied biosystems , foster city , calif .) according to the manufacturer &# 39 ; s instructions , and primers previously synthesized for sequencing of pf ama - 1 . 1 10 this resulted in the unambiguous sequence of p . falciparum fvo pf ama - 1 , that differs from the fvo ama - 1 sequence available from genbank ( accession number u84348 ) at three amino acid positions . the most notable difference is that the genbank fvo ama - 1 sequence is one amino acid shorter than any other available ama - 1 sequence , and our fvo ama - 1 sequence does not have this deletion . the sequence of gene pf ama - 1 from the fvo strain that we have established , encodes a protein of 622 amino acid residues that has six potential n - glycosylation sites . our previous experience with expressing pf ama - 1 in baculovirus / insect cells as well as with expressing pv ama - 1 in p . pastoris has shown that these n - glycosylation sites will be glycosylated in eukaryotic heterologous expression systems . as explained above , this is undesirable since native pf ama - 1 is not glycosylated . therefore , we developed a variant that exploited the lack of conservation of n - glycosylation sites in published plasmodium ama - 1 allele sequences . asn 162 was changed to lys that is present in that position in the thai - tn strain pf ama - 1 ( accession number m58547 ). thr 288 was changed to val ( present in p . vivax and p . knowlesi ama - 1 ; accession numbers y16950 and m61097 ); ser 373 was changed to asp ( present in p . knowlesi ama - 1 ); asn 422 and ser 423 were changed to asp and lys , respectively ( present in p . knowlesi , p . vivax , p . chabaudi ( accession number m25248 ) and p . fragile ama - 1 ( accession number m29898 )) and asn 499 was changed to gln ( present in p . chabaudi ama - 1 ). the nucleotide sequence with the six changed codons to delete the potential n - glycosylation sites was used to develop a synthetic gene utilizing the codon usage of p . pastoris ( nimr , london ). our previous experience with expressing pf ama - 1 in p . pastoris taught us that the high a + t content of the p . falciparum gene makes it extremely difficult to express this in p . pastoris . there are several a + t rich regions within the coding sequence that are recognized as transcription termination and / or polyadenylation sites in yeast , resulting in truncated mrnas and no protein production . the sequence of the synthetic gene was designed according to p . pastoris codon usage with the aid of the codop program . 18 this program allows codon optimization with host organism preference . it enabled design of an optimal sequence , with strategic insertion of restriction sites , and the generation of oligos of 40 nucleotides in length from both strands of the gene . the resulting set of 92 oligos was rigorously screened for the presence of potential transcription termination signals and undesirable repeats , inverted repeats , and regions of complementarity which could potentially lead to nonspecific intermolecular hybridization . the 20 nucleotide overlap between each 40 - mer primer was adjusted to give a melting temperature in the range of 62 - 68 ° c ., in order to allow subsequent use of the primers for dna sequencing . gene synthesis was by assembly polymerase chain reaction ( pcr ), using the proof - reading pfu dna polymerase , as described in reference . 18 blunt - ended pcr products corresponding to each “ half ” of the gene were cloned into pmosblue ( amersham pharmacia ) and fully sequenced on both strands before subcloning to produce the complete synthetic gene . the final product was again sequenced on both strands . the sequence of the synthetic gene fvo pf83syn is provided in fig1 . for secreted expression in p . pastoris strain km71h , we used vector ppiczα a ( invitrogen ). this vector provides an n - terminal signal sequence and a c - terminal myc epitope followed by a 6 × his tag for easy purification . gene fragments have to be cloned in frame with these sequences . primers for pcr amplification of the pf ama - 1 ectodomain were pf83a : 5 ′- ggaattccagaactactgggagcatcc - 3 ′ ( nt 73 - 92 and additional ecori restriction site , seq id no : 3 ) and pf83h : 5 ′- gctctagaatgttatcgtaacgtaggctt - 3 ′ ( complementary to nt 1615 - 1634 and additional xbai restriction site , seq id no : 4 ) or pf83a and pf83i : 5 ′- gctctagactacatgttatcgtacgtaggctt - 3 ′ ( complementary to nt 1615 - 1635 , plus stop codon plus additional xbai restriction site ; this provides the full ectodomain without myc epitope and his tag , seq id no : 5 ). a 50 μl pcr reaction contained 10 ng template dna ( fvo pf83syn ), 100 ng of each of the primers pf83a and pf83h , or pf83a and pf83i , 0 . 2 mm dntp , 5 μl 10 × pfu reaction buffer and 1 unit pfu polymerase ( promega ). amplification proceeded as follows : 1 minute , 94 ° c ., 1 minute 52 ° c ., 1 . 5 minutes 72 ° c . for 3 cycles ; 1 minute , 94 ° c ., 1 minute 60 ° c ., 1 . 5 minutes 72 ° c . for 30 cycles ; 5 minute , 72 ° c . and then stored at 4 ° c . the resulting 1578 bp pcr product was digested with ecori and xbai sequentially , and ligated into ecori / xbai digested ppiczα a in a 1 : 10 molar ratio . e . coli dh5α subcloning efficiency cells were transformed with 5 μl of the ligation mixture and plated on low salt lb plates containing 25 μg / ml zeocin and cultured overnight at 37 ° c . colonies were grown in low salt lb containing 25 μg / ml zeocin , plasmids were isolated by standard miniprep methods and analyzed by restriction enzyme digestion . one clone containing the correct insertion for each of the pcr products ( named pf4 mh for primers a and h , and pf11 - 0 for primers a and i ) was used to isolate plasmid dna for transformation of p . pastoris . the expression construct was linearized with ssti and 10 μg dna was used to transform 80 μl p . pastoris km71h cells by electroporation following the invitrogen protocols . 1 ml of 1m sorbitol was added and the cells were allowed to recover for two hours at 30 ° c . cells were then plated ( 25 , 50 , 100 , 200 μl aliquots ) on ypds ( 1 % yeast extract , 2 % peptone , 2 % dextrose , 1 m sorbitol ) agar plates containing 100 μg / ml zeocin , and incubated for 4 days at 30 ° c . colonies were picked and grown for 2 days at 30 ° c . in 10 ml of bmgy ( 1 % yeast extract , 2 % peptone , 1 . 34 % yeast nitrogen base , 1 % glycerol , 0 . 4 mg / l biotin , 0 . 1m k - phosphate ph 6 . 0 ) in 50 ml falcon tubes with vigorous shaking . cells were harvested by low - speed centrifugation , resuspended in 4 ml of bmmy ( bmgy with glycerol substituted for 0 . 5 % methanol ), and cultured for an additional 2 days . cells were harvested and the culture supernatants were analyzed for the presence of pf ama - 1 ectodomain by sds - page . gels were stained with coomassie brilliant blue . all clones analyzed expressed an equal amount of two proteins in the culture supernatant . a 50 kda molecule of thus far unknown origin , as well as an approximately 75 kda protein , which proved to be the pf ama - 1 ectodomain , with or without myc epitope and his tag ( pf4 mh and pf11 - 0 , respectively ). expression levels in these small scale cultures are estimated to be 50 mg / l . our experience with the expression of pv ama - 1 in p . pastoris suggests that this might result in levels approaching 1 g / l in optimized fermentations . no obvious degradation products were visible in the culture supernatants . culture supernatants of pf4 mh were spot blotted on nitrocellulose membranes and incubated with rat monoclonal antibody 58f8 ( recognizing a linear epitope in the n - terminal region of pf ama - 1 ), or 4g2 recognizing a conformational epitope in the ectodomain and capable of blocking parasite multiplication in vitro ) for one hour at room temperature . after incubation with goat - anti - rat igg , color was developed using nbt / bcip . only culture supernatants from the recombinant p . pastoris expressing the 75 kda protein reacted with both mabs . control culture supernatants , where the 50 kda protein , but not the 75 kda protein , was present did not react with either of the mabs . this indicates that the 75 kda protein is the pf ama - 1 ectodomain and that the secreted material is properly folded . as expected , reactivity with 4g2 was lost when the culture supernatant was reduced with β - mercaptoethanol prior to spot blotting , demonstrating the correct disulfide bond formation within the ectodomain to recreate the 4g2 epitope . purified pf4 mh ( sec 4 ) was used in a standard elisa to test reactivity with mab 4g2 and a human serum from an african endemic region . these human sera show high reactivity with conformational epitopes of ama - 1 , and hardly react with reduced ama - 1 . in this elisa , strong reactivity with 4g2 and the human serum was detected , whereas a control mab and a pool of european human serum did not react . as a positive control , similar amounts of baculovirus - produced pf ama - 1 were coated on an elisa plate and incubated with the same serum samples . similar results were obtained , although reactivity was much lower , suggesting a much better quality for the pichia pf4 mh product . in addition , rabbit sera raised against the baculovirus - produced pf ama - 1 displayed much lower titers on pf4 mh than rabbit sera raised against pf4 mh . this was not due to impurities in the pf4 mh preparation , since : 1 ) a very low reactivity of the anti - pf4 mh sera against pichia proteins was observed , and 2 ) anti - pichia antisera were only marginally reactive with contaminations in purified pf4 mh by western blotting . these results indicate that the baculovirus - produced pf ama - 1 is less immunogenic , most likely due to the relative impurity of the purified product and / or heterogeneity in folding of the product . the homogeneity of the pichia - produced pf4 mh was further evaluated by immuno - affinity chromatography , using immobilized mab 4g2 , reactive with a conformational epitope . it was found that pf4 mh quantitatively bound to the immobilized mab , demonstrating that every molecule has the proper conformation . to determine a pf ama - 1 epitope for mab 4g2 , we expressed separate domains of pf ama - 1 and combinations thereof using the same p . pastoris system as for pf4 mh . these are : pf3 mh : amino acid residues 25 - 442 ( prosequence , domains i and ii ); pf14 - 0 : residues 97 - 545 ( domains i , ii , iii ). residue 97 is the natural n - terminus of the 66 kda proteolytic product of the 83 kda pf ama - 1 . 21 we established that the parasite - inhibitory mab 4g2 is only reactive with pf3 mh , pf4 mh and pf14 - 0 , and not with any of the other proteins . this maps an epitope for 4g2 to domain i or domains i + ii . immunogenicity has been evaluated in rabbits by four immunizations of 100 microgram protein formulated in freunds complete ( 1st injection ) or freunds incomplete ( remaining injections ) adjuvant . injections were given at days 0 , 14 , 28 and 56 , and antisera obtained four weeks after the final boost were tested by elisa and immunofluorescence ( ifa ). results for pf4 mh are summarized in table 1 and ifa data from the other rabbit sera are summarized in table 2 . it is clear that all ama - 1 domains produced by us are capable of inducing high levels of antibodies that are reactive with the native parasite protein . using the same protocol , the immunogenicity of two additional fragments are evaluated . these fragments comprise : igg was purified from immunized rabbits using standard procedures , and the capacity to inhibit p . falciparum growth in vitro was evaluated . parasites at mature schizont stage were cultured in 96 - well plates in the presence of different concentrations of igg from the immunized rabbits , or of igg from control rabbits immunized with adjuvant only , or purified mab 4g2 igg . radiolabel was added after reinvasion of erythrocytes had occurred ( approximately 17 hours later ) and in vitro culture was continued for another ten hours . parasites were harvested onto glass fiber filters using a titertek cell harvester ( icn ). incorporation of [ 3 h ] hypoxanthine was determined by liquid scintillation spectrometry . parasite growth inhibition , reported as a percentage , was determined as follows : 100 −(( average cpm experimental / average cpm control control )× 100 ). the incorporation of erythrocytes alone was subtracted from all averages prior to determining the percentage inhibition . control igg was isolated from rabbits that had been immunized with adjuvant only . in this assay , mab 4g2 at 1 mg / ml gives 50 - 60 % inhibition of invasion , irrespective of the p . falciparum strain used . data for the pf4 mh - immunized rabbit iggs are given in table 1 . we used fcr3 as the homologous strain , since ama - 1 differs by only 1 amino acid residue , located in the pro - sequence , from fvo ama - 1 . nf54 was used as the heterologous strain and differs by 29 amino acids from fvo ama - 1 . total igg from rabbits immunized with pf4 mh inhibit invasion of the homologous strain up to 85 % at 1 . 5 mg / ml ( a concentration far below standard serum igg concentrations ), and of the heterologous strain up to 58 %. this indicates the presence of common , as well as strain - specific , epitopes and demonstrates the capacity of the pichia - produced pf ama - 1 ectodomain to induce potent parasite - inhibitory antibodies . pf11 - 0 . 1 has undergone a feasibility study for gmp production at a gmp production facility . pilot fermentations at 5 - 10 l scale have been performed to assess parameters that influence proteolytic degradation and yield . the conclusion was that addition of 0 . 4 mm edta to the standard fermentation medium at ph 6 . 0 , as well as methanol induction with a high cell density for a short period of 30 hours , and immediate freezing of the harvested culture supernatant until processing , are all beneficial to prevent proteolytic degradation . for purification , best results were obtained by direct binding of pf ama - 1 on an immobilized metal affinity column activated with cuso 4 ( lmac ). this step also removes proteases from pf ama - 1 resulting in an increase in stability of the partially purified product . the general conclusion of the feasibility study is that it is feasible to produce 1 gram of protein with a minimum purity of 98 % for phase i clinical testing . for mid - scale production of pf ama - 1 ectodomain , recombinant p . pastoris was cultured in 1 l baffled flasks ( 400 ml bmgy per flask ) for 48 hours at 29 - 30 ° c . under vigorous shaking . cells were harvested and resuspended in 100 ml bmmy , and then cultured for 48 hours at 29 - 30 ° c . under vigorous shaking . methanol was added to a final concentration of 0 . 5 % every 24 hours . after low - speed centrifugation , the culture supernatant was harvested . protein was precipitated with ammonium sulphate ( 70 % final concentration ) at 0 ° c ., and the precipitate was stored at 4 ° c . until use . additional proof that the secreted 75 kda protein is the pf ama - 1 ectodomain comes from purification using ni resins , since recombinant proteins produced using the ppiczα vector contain his tags that have a high affinity for ni . the ammonium sulphate precipitate of 50 ml culture supernatant was solubilized in 2 ml binding buffer ( 20 mm na phosphate ph 7 . 8 , 0 . 5 m nacl ) and loaded on an 8 ml ni - agarose column ( probond , bio - rad ) at 0 . 2 ml / minute . the column was washed at 1 ml / minute with 15 ml binding buffer , 25 ml of the same buffer ph 6 . 0 , 15 ml of the buffer ph 5 . 5 and then eluted with the same buffer at ph 4 . 0 . elution was monitored at 280 nm . the ph 4 . 0 peak fractions contained a single protein of 75 kda as determined by sds - page analysis . alternatively , the 75 kda ectodomain could be eluted with a linear 0 - 500 mm imidazole gradient in 20 mm na phosphate ph 6 . 0 , 0 . 5 m nacl . spot blotting of the peak fractions revealed strong 4g2 and 58f8 binding , indicating that the 75 kda protein is the his - tagged pf ama - 1 ectodomain . the 50 kda protein present in the culture supernatant as well as yellow - stained flavin components were present in the flow through and ph 6 . 0 wash fractions . other purification strategies are needed when the ectodomain is expressed without his tag , which might be more appropriate for clinical purposes . one way of purifying the 75 kda ectodomain pf11 - 0 away from the 50 kda protein is the use of hydroxy apatite ( hap ) 19 , 2 ° chromatography . the ammonium sulphate precipitate of a 100 ml culture supernatant was solubilized in 5 ml 10 mm napo 4 , ph 6 . 8 and loaded onto a prepacked 5 ml hap column ( cht - ii , bio - rad ) at 0 . 5 ml / minute . elution with a 20 ml gradient to 400 mm napo 4 , ph 6 . 8 at 1 ml / minute was monitored at 280 nm . two overlapping peaks were evident , the first one containing mainly the 50 kda protein , the second one mainly the pf ama - 1 ectodomain . further purification could be obtained by subsequent anion exchange chromatography of the pooled second peak fractions after diluting 1 : 10 in miliq water on a prepacked 5 ml uno q column ( bio - rad ), eluted with a linear gradient of 0 - 0 . 5 m nacl in 20 mm tris hcl ph 7 . 6 . this results in several peaks containing the remainder of the 50 kda contaminant as well as several degradation products of the ama - 1 ectodomain , and a single peak that contains pure intact ama - 1 ectodomain , as analyzed by reduced sds - page and coomassie staining . the 50 kda protein present in the culture supernatant of our recombinant p . pastoris km71h clones is not common ( information from invitrogen ). transformation of just the empty ppiczα vector into the same batch of p . pastoris km71h also yielded a 50 kda protein in the culture supernatant upon methanol induction . untransformed p . pastoris km71h does not produce this protein . we have now succeeded in preparing a new clone ( pf11 - 0 . 1 ) that only secretes the 75 kda pf ama - 1 ectodomain upon methanol induction , and that does not produce the 50 kda contaminant . this was achieved by picking a single colony of p . pastoris km71h from a freshly prepared agar plate , made from the original stock of that strain . this colony was used to start fresh cultures that were transformed with the pf11 - 0 vector , resulting in the above - described expression . purification as described under 4 . 2 will provide higher yields of pure pf ama - 1 ectodomain , since there is no need to separate the 75 kda product from a major contaminant any more , thus allowing recovery of the complete peak fraction 20 from the hap column for further anion exchange chromatography purification . 1 . collins , w . e ., et al ., protective immunity induced in squirrel monkeys with recombinant apical membrane antigen - 1 of plasmodium fragile . am . j . trop . med . hyg ., 1994 . 51 ( 6 ): p . 711 - 9 . 2 . deans , j . a . and w . c . jean , structural studies on a putative protective plasmodium knowlesi merozoite antigen . mol . biochem . parasitol ., 1987 . 26 ( 1 - 2 ): p . 155 - 66 . 3 . anders , r . f ., et al ., immunisation with recombinant ama - 1 protects mice against infection with plasmodium chabaudi . vaccine , 1998 . 16 ( 2 - 3 ): p . 240 - 7 . 4 . crewther , p . e ., et al ., protective immune responses to apical membrane antigen 1 of plasmodium chabaudi involve recognition of strain - 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2 ): p . 333 - 6 . 10 . thomas , a . w ., a . p . waters , and d . can , analysis of variation in pf83 , an erythrocytic merozoite vaccine candidate antigen of plasmodium falciparum . mol . biochem . parasitol ., 1990 . 42 ( 2 ): p . 285 - 7 . 11 . deans , j . a ., et al ., vaccination trials in rhesus monkeys with a minor , invariant , plasmodium knowlesi 66 kd merozoite antigen . parasite immunol ., 1988 . 10 ( 5 ): p . 535 - 52 . 12 . deans , j . a ., et al ., rat monoclonal antibodies which inhibit the in vitro multiplication of plasmodium knowlesi . clin . exp . immunol ., 1982 . 49 ( 2 ): p . 297 - 309 . 13 . thomas , a . w ., et al ., the fab fragments of monoclonal igg to a merozoite surface antigen inhibit plasmodium knowlesi invasion of erythrocytes . mol . biochem . parasitol ., 1984 . 13 ( 2 ): p . 187 - 99 . 14 . kocken , c . h ., et al ., precise timing of expression of a plasmodium falciparum - derived transgene in plasmodium berghei is a critical determinant of subsequent subcellular localization . j . biol . chem ., 1998 . 273 ( 24 ): p . 15119 - 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