巴斯德毕赤酵母(Pichia pastoris)表达系统综述27页PPT
巴斯德毕赤酵母(Pichia pastoris)表达系统综述28页PPT
21、要知道对好事的称颂过于夸大，也会招来人们的反感轻蔑和嫉妒。——培根 22、业精于勤，荒于嬉；行成于思，毁于随。——韩愈
23、一切节省，归根到底都归结为时间的节省。——马克思 24、意志命运往往背道而驰，决心到最后会全部推倒。——莎士比亚
25、学习是劳动，是充满思想的劳动——乌申斯基
巴斯德毕赤酵母(Pichia pastoris)表达 系统综述
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6、黄金时代是在我们的前面，而不在 我们的 后面。
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7、心急吃不了热汤圆。
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8、你可以很有个性，但某些时候请收 敛。
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9、只为成功找方法，不为失败找借口 (蹩脚 的工人 总是说 工具不 好)。
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10、只要下定决心克服恐惧，便几乎 能克服 任何恐 惧。因 为，请 记住， 除了在 脑海中 ，恐惧 无处藏 身。-- 戴尔． 卡耐基 。
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毕赤酵母表达系统的绝好综述文献共享
Heterologous protein expression in the methylotrophic yeastPichia pastorisJoan Lin Cereghino,James M.Cregg*Department of Biochemistry and Molecular Biology,Oregon Graduate Institute of Science and Technology,20000N.W.Walker Road,Beaverton,OR97006-8921,USAReceived25July1999;accepted4September1999AbstractDuring the past15years,the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins.The increasing popularity of this particular expression system can be attributed to several factors,most importantly:(1)the simplicity of techniques needed for the molecular genetic manipulation of P.pastoris and their similarity to those of Saccharomyces cerevisiae,one of the most well-characterized experimental systems in modern biology;(2)the ability of P.pastoris to produce foreign proteins at high levels,either intracellularly or extracellularly;(3)the capability of performing many eukaryotic post-translational modifications,such as glycosylation,disulfide bond formation and proteolytic processing;and(4)the availability of the expression system as a commercially available kit.In this paper,we review the P.pastoris expression system:how it was developed,how it works,and what proteins have been produced.We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.ß2000Federation of European Microbiological Societies.Published by Elsevier Science B.V.All rights reserved.Keywords:Foreign gene expression;Heterologous protein production;Methylotrophic yeast;Pichia pastoris;Alcohol oxidase1gene promoter;Protein secretionContents1.Introduction (46)1.1.Pichia pastoris as an experimental organism (46)1.2.Methanol metabolism (46)1.3.AOX1promoter (46)1.4.Molecular genetic manipulation (47)2.Construction of expression strains (47)2.1.Expression vectors (48)2.2.Alternative promoters (48)2.3.Selectable markers (48)2.4.Host strains (49)2.5.Integration of expression vectors into the P.pastoris genome (50)2.6.Generating multicopy strains (50)2.7.High cell density growth in fermenter cultures (50)3.Post-translational modi¢cation of secreted proteins (52)3.1.Secretion signal selection (52)3.2.O-Linked glycosylation (53)3.3.N-Linked glycosylation (53)4.Conclusions (53)0168-6445/00/$20.00ß2000Federation of European Microbiological Societies.Published by Elsevier Science B.V.All rights reserved.PII:S0168-6445(99)00029-7Acknowledgements .........................................................58References ...............................................................581.Introduction1.1.Pichia pastoris as an experimental organismThirty years ago,Koichi Ogata ¢rst described the ability of certain yeast species to utilize methanol as a sole source of carbon and energy [1].The methylotrophs attracted immediate attention as potential sources of single-cell pro-tein (SCP)to be marketed primarily as high-protein ani-mal feed.During the 1970s,Phillips Petroleum Company developed media and protocols for growing Pichia pastoris on methanol in continuous culture at high cell densities (s 130g l 31dry cell weight,Fig.1)[2].Unfortunately,the oil crisis of the 1970s caused a dramatic increase in the cost of methane.Concomitantly,the price of soybeans,the major alternative source of animal feed,fell.As a result,the economics of SCP production from methanol were never favorable.In the following decade,Phillips Petroleum contracted with the Salk Institute Biotechnology/Industrial Associ-ates,Inc.(SIBIA,La Jolla,CA)to develop P.pastoris as an organism for heterologous protein expression.Re-searchers at SIBIA isolated the gene and promoter for alcohol oxidase,and generated vectors,strains,and corre-sponding protocols for the molecular genetic manipulation of P.pastoris .The combination of the fermentation meth-ods developed for the SCP process and the alcohol oxidase promoter's strong,regulated expression e¡ected surpris-ingly high levels of foreign protein expression.In 1993,Phillips Petroleum sold its P.pastoris expression system patent position to Research Corporation Technologies (Tucson,AZ),the current patent holder.In addition,Phil-lips Petroleum licensed Invitrogen Corporation (Carlsbad,CA)to sell components of the system,an arrangement that continues under Research Corporation Technologies.1.2.Methanol metabolismThe conceptual basis for the P.pastoris expression sys-tem stems from the observation that some of the enzymes required for methanol metabolism are present at substan-tial levels only when cells are grown on methanol [3,4].Biochemical studies showed that methanol utilization re-quires a novel metabolic pathway involving several unique enzymes [3].The enzyme alcohol oxidase (AOX)catalyzes the ¢rst step in the methanol utilization pathway,the ox-idation of methanol to formaldehyde and hydrogen per-oxide (Fig.2).AOX is sequestered within the peroxisome along with catalase,which degrades hydrogen peroxide to oxygen and water.A portion of the formaldehyde gener-ated by AOX leaves the peroxisome and is further oxi-dized to formate and carbon dioxide by two cytoplasmic dehydrogenases,reactions that are a source of energy for cells growing on methanol.The remaining formaldehyde is assimilated to form cel-lular constituents by a cyclic pathway that starts with the condensation of formaldehyde with xylulose 5-monophos-phate,a reaction catalyzed by a third peroxisomal enzyme dihydroxyacetone synthase (DHAS).The products of this reaction,glyceraldehyde 3-phosphate and dihydroxyace-tone,leave the peroxisome and enter a cytoplasmic path-way that regenerates xylulose 5-monophosphate and,for every three cycles,one net molecule of glyceraldehyde 3-phosphate.Two of the methanol pathway enzymes,AOX and DHAS,are present at high levels in cells grown on methanol but are not detectable in cells grown on most other carbon sources (e.g.,glucose,glycerol,or ethanol).In cells fed methanol at growth-limiting rates in fermenter cultures,AOX levels are dramatically induced,constitut-ing s 30%of total soluble protein [5,6].1.3.AOX1promoterThere are two genes that encode alcohol oxidase in P.pastoris :AOX1and AOX2;AOX1is responsible for a vast majority of alcohol oxidase activity in the cell[7^9].Fig.1.High cell density culture of P.pastoris .The centrifuge bottle on the left shows a P.pastoris culture grown in a £ask to a density of 1OD 600unit.The bottle on the right contains a sample of the strain grown in a fermenter to a density of 130g l 31dry cell weight (V 500OD 600units).J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^6646Expression of the AOX1gene is controlled at the level of transcription [7^9].In methanol-grown cells,V 5%of poly(A) RNA is from AOX1;however,in cells grown on most other carbon sources,AOX1message is undetect-able [10].The regulation of the AOX1gene appears to involve two mechanisms:a repression/derepression mech-anism plus an induction mechanism,similar to the regu-lation of the Saccharomyces cerevisiae GAL1gene.Unlike GAL1regulation,the absence of a repressing carbon source,such as glucose in the medium,does not result in substantial transcription of AOX1.The presence of meth-anol is essential to induce high levels of transcription [7].1.4.Molecular genetic manipulationTechniques required for the molecular genetic manipu-lation of P.pastoris ,such as DNA-mediated transforma-tion,gene targeting,gene replacement,and cloning by functional complementation,are similar to those described for S.cerevisiae .P.pastoris can be transformed by electro-poration,a spheroplast generation method,or whole cell methods such as those involving lithium chloride and polyethylene glycol 1000[11^14].As in S.cerevisiae ,P.pas-toris exhibits a propensity for homologous recombination between genomic and arti¢cially introduced DNAs.Cleav-age of a P.pastoris vector within a sequence shared by the host genome stimulates homologous recombination events that e¤ciently target integration of the vector to that ge-nomic locus [15].Gene replacements occur at lower fre-quencies than those observed in S.cerevisiae and appear to require longer terminal £anking sequences to e¤ciently direct integration [14].P.pastoris is a homothallic ascomycetous yeast that can also be manipulated by classical genetic methods [10,16].Unlike homothallic strains of S.cerevisiae ,which are dip-loid,P.pastoris remains haploid unless forced to mate.Strains with complementary markers can be mated by subjecting them to a nitrogen-limited medium.After 1day on this medium,cells are shifted to a standardminimal medium supplemented with nutrients designed to select for complementing diploid cells (not self-mated or non-mated parental cells).The resulting diploids are stable as long as they are not subjected to nutritional stress.To obtain spore products,diploids are returned to the nitrogen-limited medium,which stimulates them to proceed through meiosis and sporulation.Spore products are handled by random spore techniques rather than micromanipulation,since P.pastoris asci are small and di¤cult to dissect.Yet most standard classical genetic ma-nipulations,including mutant isolation,complementation analysis,backcrossing,strain construction,and spore analysis,can be accomplished.2.Construction of expression strainsExpression of any foreign gene in P.pastoris requires three basic steps:(1)the insertion of the gene into an expression vector;(2)introduction of the expression vec-tor into the P.pastoris genome;and (3)examination of potential expression strains for the foreign gene product.A variety of P.pastoris expression vectors and host strains are available.A generalized diagram of an expression vec-tor and a list of possible vector components are shown in Fig.3and Table 1,respectively.More detailed informa-tion on vectors and strains can be found elsewhere [17,18].In addition,the DNA sequence of many vectors can be found at the Invitrogen website ().Table 2shows a list of commonly used P.pastoris hoststrains.Fig.2.The methanol pathway in P.pastoris .1,alcohol oxidase;2,cata-lase;3,formaldehyde dehydrogenase;4,formate dehydrogenase,5,di-hydroxyacetone synthase;6,dihydroxyacetone kinase;7,fructose 1,6-bi-phosphate aldolase;8,fructose1,6-bisphosphatase.Fig.3.General diagram of a P.pastoris expression vector.YFG,`Your Favorite Gene;'*,sites for cassette ampli¢cation.J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^66472.1.Expression vectorsAll expression vectors have been designed as Escherichia coli/P.pastoris shuttle vectors,containing an origin of rep-lication for plasmid maintenance in E.coli and markers functional in one or both organisms.Most expression vec-tors have an expression cassette composed of a 0.9-kb fragment from AOX1composed of the 5P promoter se-quences and a second short AOX1-derived fragment with sequences required for transcription termination [19].Be-tween the promoter and terminator sequences is a site or multiple cloning site (MCS)for insertion of the foreign coding sequence.In the native AOX1gene,the alcohol oxidase open reading frame (ORF)is preceded by an un-usually long 5P untranslated region (116nt)[8].Generally,the best expression results are obtained when the ¢rst ATG of the heterologous coding sequence is inserted as close as possible to the position of the AOX1ATG.This position coincides with the ¢rst restriction site in most MCSs.In addition,for secretion of foreign proteins,vec-tors are available where in-frame fusions of foreign pro-teins and the secretion signals of P.pastoris acid phospha-tase (PHO1)or S.cerevisiae K -mating factor (K -MF)can be generated.2.2.Alternative promotersAlthough the AOX1promoter has been successfully used to express numerous foreign genes,there are circum-stances in which this promoter may not be suitable.For example,the use of methanol to induce gene expression may not be appropriate for the production of food prod-ucts since methane,a petroleum-related compound,is one source of methanol.Also,methanol is a potential ¢re haz-ard,especially in quantities needed for large-scale fermen-tations.Therefore,promoters that are not induced by methanol are attractive for expression of certain genes.Alternative promoters to the AOX1promoter are the P.pastoris GAP ,FLD1,PEX8,and YPT1promoters.2.2.1.P GAPBoth northern and reporter activation results indicate that the P.pastoris glyceraldehyde 3-phosphate dehydro-genase (GAP )gene promoter provides strong constitutive expression on glucose at a level comparable to that seen with the AOX1promoter [20].GAP promoter activity lev-els in glycerol-and methanol-grown cells are approxi-mately two-thirds and one-third of the level observed for glucose,respectively.The advantage of using the GAP promoter is that methanol is not required for induction,nor is it necessary to shift cultures from one carbon source to another,making strain growth more straightforward.However,since the GAP promoter is constitutively ex-pressed,it is not a good choice for the production of proteins that are toxic to the yeast.2.2.2.P FLD1The FLD1gene encodes a glutathione-dependent form-aldehyde dehydrogenase,a key enzyme required for the metabolism of certain methylated amines as nitrogen sour-ces and methanol as a carbon source [21].The FLD1pro-moter can be induced with either methanol as a sole car-bon source (and ammonium sulfate as a nitrogen source)or methylamine as a sole nitrogen source (and glucose as a carbon source).After induction with either methanol or methylamine,P FLD1is able to express levels of a L -lacta-mase reporter gene similar to those obtained with metha-nol induction from the AOX1promoter.The FLD1pro-moter o¡ers the £exibility to induce high levels of expression using either methanol or methylamine,an inex-pensive nontoxic nitrogen source.2.2.3.P PEX8,P YPT1For some applications,the AOX1,GAP ,and FLD1promoters may be too strong,expressing genes at too high a level.There is evidence that,for certain foreign genes,the high level of expression from P AOX1may over-whelm the post-translational machinery of the cell,causing a signi¢cant proportion of foreign protein to be misfolded,unprocessed,or mislocalized [22,23].For these and other applications,moderately expressing promoters are desir-able.Toward this end,the P.pastoris PEX8and YPT1promoters may be of use.The PEX8gene encodes a per-oxisomal matrix protein that is essential for peroxisome biogenesis [24].It is expressed at a low but signi¢cant level on glucose and is induced modestly when cells are shifted to methanol.The YPT1gene encodes a GTPase involved in secretion,and its promoter provides a low but constit-utive level of expression in media containing either glu-cose,methanol,or mannitol as carbon sources [25].2.3.Selectable markersAlthough classical and molecular genetic techniques are generally well-developed for P.pastoris ,few selectable marker genes have been described for the molecular genet-ic manipulation of the yeast.Existing markers are limited to the biosynthetic pathway genes HIS4from either P.pastoris or S.cerevisiae ,ARG4from S.cerevisiae ,and the Sh ble gene from Streptoalloteichus hindustanus which confers resistance to the bleomycin-related drug zeocin [11,26,27].The stable expression of human type III colla-gen illustrates the need for multiple selectable markers inTable 1Relevant components of vectors used for protein expression in P.past-orisSecretion signals none,PHO1,K -MF,SUC2,PHA-EMarker genes ADE1,ARG4,G418,HIS4,URA3,Zeo r PromotersAOX1,GAP,FLD1,PEX8,YPT1See text for explanation of di¡erent elements.J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^6648P.pastoris[28].The production of collagen requires the coexpression of prolyl4-hydroxylase,a central enzyme in the synthesis and assembly of trimeric collagen.Since prol-yl4-hydroxylase is an K2L2tetramer,the L subunit of which is protein disul¢de isomerase(PDI),three markers ^Arg,His,and zeocin resistance^were necessary to co-express all three polypeptides in the same P.pastoris strain.Recently,a new set of biosynthetic markers has been isolated and characterized:the P.pastoris ADE1(PR-ami-doimidazolesuccinocarboxamide synthase),ARG4(argini-nosuccinate lyase),and URA3(orotidine5P-phosphate de-carboxylase)genes[29].Each of these selectable markers has been incorporated into expression vectors.In addition, a series of host strains containing all possible combina-tions of ade1,arg4,his4,and ura3auxotrophies has been generated(Table2).2.4.Host strainsAll P.pastoris expression strains are derived from NRRL-Y11430(Northern Regional Research Laborato-ries,Peoria,IL).Most have one or more auxotrophic mu-tations which allow for selection of expression vectors containing the appropriate selectable marker gene upon transformation.Prior to transformation,all of these strains grow on complex media but require supplementa-tion with the appropriate nutrient(s)for growth on mini-mal media.2.4.1.Methanol utilization phenotypeMost P.pastoris host strains grow on methanol at the wild-type rate(Mut ,methanol utilization plus pheno-type).However,two other types of host strains are avail-able which vary with regard to their ability to utilize meth-anol because of deletions in one or both AOX genes. Strains with AOX mutations are sometimes better pro-ducers of foreign proteins than wild-type strains[30^32]. Additionally,these strains do not require the large amounts of methanol routinely used for large-scale fer-mentations of Mut strains.KM71(his4arg4aox1v:: SARG4)is a strain where AOX1has been partially deleted and replaced with the S.cerevisiae ARG4gene[15].Since the strain must rely on the weaker AOX2for methanol metabolism,it grows slowly on this carbon source (Mut s,methanol utilization slow phenotype).Another strain,MC100-3(his4arg4aox1v::SARG4aox2v:: Phis4),is deleted for both AOX genes and is totally unable to grow on methanol(Mut3,methanol utilization minus phenotype)[9].All of these strains,even the Mut3strain, retain the ability to induce expression at high levels from the AOX1promoter[32].2.4.2.Protease-de¢cient host strainsSeveral protease-de¢cient strains^SMD1163(his4pep4 prb1),SMD1165(his4prb1),and SMD1168(his4pep4)^ have been shown to be e¡ective in reducing degradation of some foreign proteins[23,33].This is especially noticeable in fermenter cultures,because the combination of high cellTable2P.pastoris host strainsStrain Genotype Reference Auxotrophic strainsY-11430wild-type NRRL aGS115his4[11]GS190arg4[16]JC220ade1[16]JC254ura3[16]GS200arg4his4[11]JC227ade1arg4[29]JC304ade1his4[29]JC305ade1ura3[29]JC306arg4ura3[29]JC307his4ura3[29]JC300ade1arg4his4[29]JC301ade1his4ura3[29]JC302ade1arg4ura3[29]JC303arg4his4ura3[29]JC308ade1arg4his4ura3[29] Protease-de¢cient strainsKM71v aox1::SARG4his4arg4[7]MC100-3v aox1::SARG4v aox2::Phis4his4arg4[9]SMD1168v pep4::URA3his4ura3[38]SMD1165prb1his4[38]SMD1163pep4prb1his4[38]SMD1168kex1::SUC2v pep4::URA3v kex1::SUC2his4ura3[34]a Northern Regional Research Laboratories,Peoria,IL.J.L.Cereghino,J.M.Cregg/FEMS Microbiology Reviews24(2000)45^6649density and lysis of a small percentage of cells results in a relatively high concentration of these vacuolar proteases. An additional protease-de¢cient strain SMD1168v pe-p4::URA3v kex1::SUC2his4ura3was recently devel-oped to inhibit proteolysis of murine and human endo-statin.Kex1protease can cleave carboxy-terminal lysines and arginines.Therefore,the deletion strain was generated to inhibit carboxy-terminal proteolysis.After40h of fer-mentation,puri¢cation of intact endostatin was achieved [34].Unfortunately,these protease-de¢cient cells are not as vigorous as wild-type strains with respect to PEP4.In addition to lower viability,they possess a slower growth rate and are more di¤cult to transform.Therefore,the use of protease-de¢cient strains is only recommended in situa-tions where other measures to reduce proteolysis have yielded unsatisfactory results.2.5.Integration of expression vectors into the P.pastorisgenomeExpression vectors are integrated into the P.pastoris genome to maximize the stability of expression strains. This can be done in two ways.The simplest way is to restrict the vector at a unique site in either the marker gene(e.g.,HIS4)or the AOX1promoter fragment and then to transform it into the appropriate auxotrophic mu-tant.The free DNA termini stimulate homologous recom-bination events that result in single crossover-type integra-tion events into these loci at high frequencies(50^80%of His transformants).The remaining transformants have undergone gene conversion events in which only the marker gene from the vector has integrated into the mu-tant host locus without other vector sequences. Alternatively,certain P.pastoris expression vectors can be digested in such a way that the expression cassette and marker gene are released,£anked by5P and3P AOX1 sequences.Approximately10^20%of transformation events are the result of a gene replacement event in which the AOX1gene is deleted and replaced by the expression cassette and marker gene.This disruption of the AOX1 gene forces these strains to rely on the transcriptionally weaker AOX2gene for growth on methanol[31],and,as a result,these strains have a Mut s phenotype.These gene replacement strains are easily identi¢ed among trans-formed colonies by replica-plating them to methanol and selecting those with reduced ability to grow on methanol. As mentioned previously,the potential advantage of Mut s strains is that they utilize less methanol and sometimes express higher levels of foreign protein than wild-type (Mut )strains,especially in shake-£ask cultures[15].2.6.Generating multicopy strainsOptimization of protein expression often,but not al-ways,includes the isolation of multicopy expression strains.A strain that contains multiple integrated copies of an expression cassette can sometimes yield more heter-ologous protein than single-copy strains[22,35].Three approaches lead reliably to multicopy expression strains in P.pastoris.As shown in Fig.4,the¢rst ap-proach involves constructing a vector with multiple head-to-tail copies of an expression cassette[23].The key to generating this construction is a vector which has an expression cassette£anked by restriction sites which have complementary termini(e.g.,Bam HI-Bgl II,Sal I-Xho I combinations).The process of repeated cleavage and reinsertion results in the generation of a series of vectors that contain increasing numbers of expression cas-settes.A particular advantage to this approach,especially in the production of human pharmaceuticals,is that the precise number of expression cassettes is known and can be recovered for direct veri¢cation by DNA sequencing.A second method utilizes expression vectors that con-tain the P.pastoris HIS4and the bacterial Tn903kan r genes.The bacterial kanamycin resistance gene also con-fers resistance to the related eukaryotic antibiotic G418 [36].The level of G418resistance can be roughly corre-lated to vector copy number.P.pastoris must¢rst be transformed to His prototrophy;then multicopy trans-formants are screened by replica-plating to plates contain-ing G418.This method results in a subset of colonies enriched for those containing multiple expression vector copies.However,the vector copy number varies greatly; thus,a signi¢cant number(50^100)of transformants must be subjected to further analysis of copy number and ex-pression level.By this approach,strains carrying up to30 copies of an expression cassette have been isolated[35].A third approach to constructing multicopy strains in-volves the use of a vector with the bacterial Sh ble gene, which confers resistance to the antibiotic zeocin[27].Un-like G418selection,strains transformed with expression cassettes containing the zeocin marker can be selected di-rectly by resistance to the drug.Additionally,populations of transformants can be enriched for multicopy expression cassette strains simply by plating on increased concentra-tions of zeocin in the selection plates.Also,because the Sh ble gene can serve as a selectable marker in both bacteria and yeast,these expression vectors are compact and con-venient to use.However,as with the G418selection,most transformants resistant to high levels of zeocin do not contain multiple vector copies,and numerous transform-ants must be screened for ones that do.2.7.High cell density growth in fermenter culturesP.pastoris is a poor fermenter,a major advantage rel-ative to S.cerevisiae.In high cell density cultures,ethanol (the product of S.cerevisiae fermentation)rapidly builds to toxic levels which limit further growth and foreign protein production.With its preference for respiratory growth,P.pastoris can be cultured at extremely high den-J.L.Cereghino,J.M.Cregg/FEMS Microbiology Reviews24(2000)45^66 50sities (500OD 600U ml 31)in the controlled environment of the fermenter with little risk of `pickling'itself.Fermenta-tion growth is especially important for secreted proteins,as the concentration of product in the medium is roughly proportional to the concentration of cells in culture.An-other positive aspect of growing P.pastoris in fermenter cultures is that the level of transcription initiated from the AOX1promoter can be 3^5times greater in cells fed meth-anol at growth-limiting rates compared to cells grown in excess methanol.Thus,even for intracellularly expressed proteins,product yields are signi¢cantly higher from fer-menter cultured cells.Also,methanol metabolism utilizes oxygen at a high rate,and expression of foreign genes is negatively a¡ected by oxygen limitation.Only in the con-trolled environment of a fermenter is it feasible to monitor and adjust oxygen levels in the culture medium.A hallmark of the P.pastoris system is the ease with which expression strains scale-up from shake-£ask to high-density fermenter cultures.Although some foreign pro-teins have expressed well in shake-£ask cultures,expres-sion levels are typically low compared to fermenter cul-tures.Considerable e¡ort has gone into the optimization of heterologous protein expression techniques,and de-tailed fed-batch and continuous culture protocols are available [23,37^39].In general,strains are grown initially in a de¢ned medium containing glycerol as its carbon source.During this time,biomass accumulates but heter-ologous gene expression is fully repressed.Upon depletion of glycerol,a transition phase is initiated in which addi-tional glycerol is fed to the culture at a growth-limiting rate.Finally,methanol or a mixture of glycerol and meth-anol is fed to the culture to induce expression.Thecon-Fig.4.Scheme for construction of vectors with multiple copies of a foreign gene expression cassette (from [22]).J.L.Cereghino,J.M.Cregg /FEMS Microbiology Reviews 24(2000)45^6651centration of foreign protein is monitored in the culture to determine time of harvest.The growth conditions for P.pastoris are ideal for large-scale production of heterologous protein,because the me-dium components are inexpensive and de¢ned,consisting of pure carbon sources(glycerol and methanol),biotin, salts,trace elements,and water.This medium is free of unde¢ned ingredients that can be sources of pyrogens or toxins and is therefore compatible with the production of human pharmaceuticals.Also,since P.pastoris is cultured in media with a relatively low pH and methanol,it is less likely to become contaminated by most other microorgan-isms.3.Post-translational modi¢cation of secreted proteinsA major advantage of P.pastoris over bacterial expres-sion systems is that the yeast has the potential to perform many of the post-translational modi¢cations typically as-sociated with higher eukaryotes,such as processing of sig-nal sequences(both pre and prepro type),folding,disul¢de bridge formation,certain types of lipid addition,and O-and N-linked glycosylation.3.1.Secretion signal selectionForeign proteins expressed in P.pastoris can be pro-duced either intracellularly or extracellularly.Because this yeast secretes only low levels of endogenous proteins, the secreted heterologous protein constitutes the vast ma-jority of total protein in the medium(Fig.5).Therefore, directing a heterologous protein to the culture medium can serve as a substantial¢rst step in puri¢cation.However, due to protein stability and folding requirements,the op-tion of secretion is usually reserved for foreign proteins that are normally secreted by their native hosts.In many cases,researchers simply need to take advantage of the pre-made expression cassettes available from Invitrogen. Using selected P.pastoris vectors,researchers can clone a foreign gene in frame with sequences encoding either the native signal,the S.cerevisiae K-factor prepro peptide, or the P.pastoris acid phosphatase(PHO1)signal. Although several di¡erent secretion signal sequences, including the native secretion signal present on heterolo-gous proteins,have been used successfully,results have been variable.The S.cerevisiae K-factor prepro peptide has been used with the most success.This signal sequence consists of a19-amino acid signal(pre)sequence followed by a66-residue(pro)sequence containing three consensus N-linked glycosylation sites and a dibasic Kex2endopep-tidase processing site[40].The processing of this signal sequence involves three steps.The¢rst is the removal of the pre signal by signal peptidase in the endoplasmic retic-ulum.Second,Kex2endopeptidase cleaves between Arg-Lys of the pro leader sequence.This is rapidly followed by cleavage of Glu-Ala repeats by the Ste13protein[41].The e¤ciency of this process can be a¡ected by the surround-ing amino acid sequence.For instance,the cleavage e¤-ciencies of both Kex2and Ste13proteins can be in£uenced by the close proximity of proline residues.In addition,the tertiary structure formed by a foreign protein may protect cleavage sites from their respective proteases.The S.cerevisiae K-MF prepro signal sequence is the classical and most widely used secretion signal(see Table 3,expressed proteins).In some cases,it is a better secre-tion signal for expression in P.pastoris than the leader sequence of the native heterologous protein.In a study concerning the expression of the industrial lipase Lip1 from Candida rugosa,the e¡ect of heterologous leader sequences on expression and secretion was investigated [42].It was found that the native Lip1p leader sequence allowed for secretion but somehow hampered expression. Either the K-factor pre or prepro signal was adequate for both secretion and expression,but the highest level of lipase secretion was from a clone with the full prepro sequence.This clone produced two species of secreted pro-tein.A small percentage was correctly processed to the mature protein.However,a majority of the product con-tained four additional N-terminal amino acids.Variability in the amino terminus is commonly seen with heterologous proteins secreted by P.pastoris using the K-factor prepro leader.In some cases,the standard K-MF or PHO1secretion signals have not worked,so synthetic leaders have been created.Martinez-Ruiz et al.[43]made mutations in the native leader to reconstruct a more e¤cient Kex2p recog-nition motif(Lys-Arg).This aided in secretion of the ri-bosome-inactivation protein K-sarcin from the mold As-pergillus giganteus.Another more drastic solution was to create an entirely synthetic prepro leader.For the expres-sion of human insulin,a synthetic leader and spacer se-quence was found to improve secretion and protein yield[44].Fig.5.Secreted expression of human serum albumin.7.5%SDS-PAGE of25-W l sample of culture supernatant from a P.pastoris strain(GS-HSA#4141)expressing human serum albumin.Cells were induced in BMMY(bu¡ered methanol-complex medium)for0,12,24,48,and ne M contains molecular mass markers(kDa).J.L.Cereghino,J.M.Cregg/FEMS Microbiology Reviews24(2000)45^66 52。
毕赤酵母表达系统资料整理
毕赤酵母表达零碎之相礼和热创作Mut+和Muts毕赤酵母中有两个基因编码醇氧化酶——AOX1及AOX2，细胞中大多数的醇氧化酶是AOX1基因产品，甲醇可紧密调理、诱导AOX1基因的高程度表达，较典型的是占可溶性蛋白的30%以上.AOX1基因调控分两步：抑制/往抑制机制加诱导机制.简单来说，在含葡萄糖的培育基中，即便加入诱导物甲醇转录仍受抑制.为此，用甲醇进行优化诱导时，引荐在甘油培育基中培育.留意即便在甘油中生长(往抑制)时，仍缺乏以使AOX1基因达到最低程度的表达，诱导物甲醇是AOX1基因可辨表达程度所必须的.AOX1基因已被分离，含AOX1启动子的质粒可用来促进编码外源蛋白的目的基因的表达.AOX2基因与AOX1基因有97%的同源性，但在甲醇中带AOX2基因的菌株比带AOX1基因菌株慢得多，经过这种甲醇利用缓慢表型可分离Muts菌株.在YPD(酵母膏、蛋白胨、葡萄糖)培育基中，不管是Mut+还是Muts其在对数期增殖一倍的工夫大约为2h.Mut+和Muts菌株在没有甲醇存在的状况下生长速率是一样的，存在甲醇的状况下，Mut+在对数期增殖一倍的工夫大约为4至6个小时，Muts在对数期增殖一倍的工夫大约为18个小时.菌株GS115、X-33、KM71和SMD1168的区别GS115、KM71和SMD1168等是用于表达外源蛋白的毕赤酵母受体菌，与酿酒酵母相比，毕赤酵母不会使蛋白过糖基化，糖基化后有利于蛋白的溶解或构成正确的折叠结构.GS115、KM71、SMD1168在组氨酸脱氢酶位点(His4)有渐变，是组氨酸缺陷型，假如表达载体上携带有组氨酸基因，可抵偿宿主菌的组氨酸缺陷，因而可以在不含组氨酸的培育基上挑选转化子.这些受体菌自发渐变成组氨酸野生型的概率一样平常低于10-8.GS115表型为Mut+，重组表达载体转化GS115后，长出的转化子可能是Mut+，也可能是Muts(载体取代AXO1基因)，可以在MM和MD培育基上鉴定表型.SMD1168和GS115类似，但SMD1168基因组中的Pep4基因发生渐变，是蛋白酶缺陷型，可降低蛋白酶对外源蛋白的降解作用.其中X-33由于是野生型，因而耐受性比较好，假如担心转化率的话可以考虑这种酵母菌，而X33与GS115一样都是属于MUT＋表示型，也就是说可以在含甲醇的培育基中快速生长，但是听说会对外源基因表达有影响，KM71的亲本菌在精氨酸琥珀酸裂解酶基因(arg4)有渐变，在不含精氨酸的培育基中不克不及生长.用野生型ARG4基因(约2kb)拔出到克隆的野生型AOX1基因的BamHI(AOX1基因15/16密码子)及SalI(AOX1基因227/228密码子)位点，取代了AOX1基因16-227密码子，此结构转化至KM71亲本菌(arg4his4)中，分离发生KM71 MutsArg+His-菌株，Arg+转化子遗传分析表现野生型AOX1被aox1::ARG4结构所取代，以是KM71全部转化子都是Muts表型.AOX1位点没有被完全缺失，理论上可用你的目的结构经过基因取代方法更换aox1::ARG4结构，这样重组菌株的表型是His+MutsArg-，这意味偏重组菌株生长时需精氨酸.但仅添加精氨酸其实不克不及完全缓和arg4渐变的影响，arg4菌株在含精氨酸的最小培育基中不克不及很好地生长.因而不引荐在KM71中经过取代aox1::ARG4结构来获得His＋转化子.一样平常来说，假如是胞内表达，应尽量用Muts细胞，这样得到的蛋白产品中醇氧化酶蛋白量较少而目的蛋白量绝对较多，使卑鄙纯化更易进行.而对于分泌蛋白的表达，无论是甲醇利用慢(Muts)还是甲醇利用快(Mut+)的细胞都可运用.基因重组Pichia.pastoris酵母菌体内无自然质粒，以是表达载体需与宿主染色体发生同源重组，将外源基因表达框架整合于染色体中以完成外源基因的表达，包含启动子、外源基因克隆位点、停止序列、挑选标识表记标帜等.细菌内同源重组被以为是重组质粒构建过程的难点，由于未线性化的环状质粒之间发生同源重组的几率非常低，以是重组转移载体必须用特定的限定性内切酶进行线性化处理.这种处理的目的是防止随机拔出重组时质粒在功能区断开，形成目的基因表达失活，让同源重组以指定的方式发生.表达载体次要分为以下几类：(1)胞内表达载体次要有pHIL-D2、pA0815、pPIC3K、pPICZ、pHWO10，pGAPZ、pGAPZa(Invitrogen)等.该类载体可以将目的基因表达在胞内，可以防止毕赤酵母的糖基化，次要得当于那些不克不及被糖基化相关基因的表达；(2)分泌型表达载体次要有pPIC9、pHIL-S1、pPICZα、pYAM75P等.由于毕赤酵母本人的泌内源蛋白非常少，将外源蛋白分泌到胞外，非常有利于目的蛋白质的纯化及积存.经常运用的分泌的信号序列次要是由89个氨基酸组成的α交配因子(α-factor)的引导；(3)多拷贝拔出表达载体如pPIC9K，pPIC3.5K.在某些状况下，毕赤酵母中重组基因多拷贝整合可添加所需蛋白的表达量.该载体均可用于在体内(pPIC3.5K, pPIC9K)或体外(pAO815)发生并分离多拷贝拔出，同时可检测添加重组基因的拷贝数能否添加蛋白表达量.体内整合可经过高遗传霉素抗性挑选可能的多拷贝拔出，而体外整合可经过连接发生外源基因的串联拔出.在GS115中挑选His+Mut+转化子：用SalI或StuI线性化质粒转化GS115后，大多在His4位点上发生重组，大多数转化子是Mut+表型；但是由于质粒含有AOX1基因序列，有可能在AOX1位点发生重组，毁坏野生型AOX1基因，发生His+Muts转化子，则必要在MD及MM平板上检测可证明His+ Mut+转化子.毕赤酵母表达经常运用培育基10×YNB(13.4%的无氨基酸酵母氮源)，134gYNB固体溶于1L蒸馏水，过滤灭菌，4℃保管.YPD完全培育基：酵母提取物10 g/L，蛋白胨20 g/L，葡萄糖20 g/L(固体培育基含1.5%琼脂).转化培育基RDB：每100mL加入山梨醇18g(186 g/L)，琼脂糖2g(20g/L)121℃灭菌20分钟，然后待温度降至60℃当前在超净台上加入10×YNB 10mL(13.4 g/L)，10×葡萄糖10mL(20 g/L)，500×生物素0.2mL(4×10-4g/L)，100×AA 1mL.混匀，倒平板(灭菌时只加入80ml水即可).选择培育基MD(最小葡萄糖)：配100mL，向80mL水中加入琼脂糖2g(20 g/L)121℃灭菌20分钟，待温度降至60℃当前在超净台上加入10×YNB 10mL(13.4 g/L)，10×葡萄糖10mL(20 g/L)，500×生物素0.2mL(4×10-4g/L).选择培育基MM(最小甲醇)：配100mL，向90mL水中加入琼脂糖2g(20 g/L)121℃灭菌20分钟，待温度降至60℃当前在超净台上加入10×YNB 10mL(13.4 g/L)，500×生物素0.2mL(4×10-4g/L)，0.5mL甲醇(0.5%).诱导表达培育基BMGY：配1L，酵母提取物10 g/L，蛋白胨20 g/L，3g/L K2HPO4，11.8g/L KH2PO4，加水至890mL，121℃灭菌20分钟，然后待温度降至60℃当前在超净台上加入10×YNB 100mL(13.4 g/L)，500×生物素1mL(4×10-4g/L)，甘油10mL.诱导表达培育基BMMY：酵母提取物10g/L，蛋白胨20 g/L，3g/LK2HPO4，11.8g/L KH2PO4，加水至895mL，121℃灭菌20分钟，然后待温度降至60℃当前在超净台上加入100×YNB 100mL(13.4 g/L)，500×生物素1mL(4×10-4g/L)，甲醇5mL.BMGY/BMMY含酵母浸出物及蛋白胨，可波动分泌蛋白，制止或减少分泌蛋白的分解.假如目的蛋白对中性PH蛋白酶敏感的话，可在无缓冲培育基(MGY、MM)中表达.假如没有证据证明你的分泌蛋白对中性PH值蛋白酶敏感，建议开始表达时用BMMY.假如表达蛋白降解了，测验考试在无缓冲培育基中进行表达.假如以上条件仍不克不及无效防止蛋白降解，可将基因转入SMD1168中，该菌株表型是his4pep4，缺失了蛋白酶，转化与表达程序与GS115相反，也可用于大规模发酵.用考马斯亮蓝G-250测蛋白含量。
巴斯德毕赤酵母表达系统
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毕赤酵母表达蛋白步骤
毕赤酵母表达蛋白步骤一、引言毕赤酵母（Pichia pastoris）是一种常用的真菌表达系统，被广泛应用于蛋白质的表达和生物技术研究中。
其优势包括高表达水平、易于培养和操作、能够正确折叠复杂蛋白等。
本文将介绍毕赤酵母表达蛋白的步骤。
二、构建表达载体毕赤酵母表达系统的关键是表达载体的构建。
首先，需要选择适合的表达载体，常用的有pPIC6、pPICZα等。
然后，在载体上选择合适的启动子和信号序列，以确保蛋白质能够被正确表达和分泌。
同时，还需要在表达载体上加入选择标记，如His标签、FLAG标签等，以便后续的蛋白质纯化和检测。
三、转化毕赤酵母将构建好的表达载体转化入毕赤酵母中，使其成为表达宿主。
转化方法包括电击转化、化学转化等。
其中，电击转化是常用的方法，通过电击脉冲使毕赤酵母细胞膜发生破裂，使表达载体进入细胞内。
转化后，将细胞培养在选择性培养基上，筛选出带有表达载体的毕赤酵母克隆。
四、表达蛋白经过转化筛选后，得到含有目标蛋白表达载体的毕赤酵母克隆。
接下来，需要将克隆进行培养，在适当的条件下诱导蛋白的表达。
常用的诱导剂包括甲醇、巯基乙醇等，通过加入适量的诱导剂，可以使目标蛋白得到高效表达。
五、蛋白纯化在蛋白表达后，需要进行蛋白纯化，以获得纯度较高的目标蛋白。
常用的纯化方法包括亲和层析、离子交换层析、凝胶过滤层析等。
在选择纯化方法时，需要根据目标蛋白的性质和需求进行合理选择。
同时，可以利用加入的选择标记，如His标签，通过亲和层析纯化进行快速高效的纯化。
六、蛋白鉴定和功能分析蛋白纯化后，需要进行蛋白的鉴定和功能分析。
常用的鉴定方法包括SDS-PAGE、Western blot等，可以确定蛋白的分子量和纯度。
功能分析则可以通过生物学实验来进行，如酶活测定、结合实验等，以验证目标蛋白的功能。
七、应用和展望毕赤酵母表达系统在生物技术和蛋白质研究领域有着广泛的应用。
通过该系统，可以高效表达各种蛋白，包括抗体、酶和重组蛋白等。
pichia_pastoris表达策略
.2 巴斯德毕赤酵母表达外源蛋白的降解机理及其控制策略1.2.1 巴斯德毕赤酵母表达外源蛋白的降解机理在外源蛋白的表达过程中，宿主菌毕赤酵母的胞内和胞外均有一定量的蛋白酶的表达，因此，不论是胞内表达亦或是分泌表达，大多数外源蛋白均面临着被降解的问题，这也是影响表达量的一个重要因素，同时，还增加了纯化目的蛋白的难度。
近年来，蛋白酶的研究是P.pastoris表达系统一个重点和热点。
越来越多的蛋白酶的遗传背景和生理生化性质得到深入的研究[52; 53]。
P.pastoris能根据细胞生长环境（碳源的改变以及细胞或细胞器的胁迫）来调整自身酶系，以合成与降解不同的蛋白和细胞器，液泡是蛋白质降解最主要的场所[54]，另一降解场所是细胞基质蛋白酶体中。
但是，对于外源蛋白来说，其降解常在表达和分离纯化的第一步，主要是由培养基中胞外蛋白酶，细胞外膜结合蛋白酶（cell-bound proteases）[55]和细胞自噬或裂解释放的胞内蛋白酶降解的[5]。
胞内蛋白酶主要涉及降解蛋白质前体产生活性蛋白；切除转运出膜后的蛋白质信号肽；使调控蛋白失活；降解变异或不需要的蛋白质；提供营养，前体和能量。
胞外蛋白酶分泌较少，主要降解部分蛋白质提供氨基酸和多肽等营养[22]。
根据蛋白酶的分泌和作用地点，P.pastoris的胞内蛋白酶可以分为三种类别，即液泡蛋白酶（vacuolar proteases），细胞基质蛋白酶体（the cytosolic proteosome）以及分泌途径的蛋白酶（proteases located along the secretory pathway）[52; 56]。
表1.2 毕赤酵母液泡蛋白酶和分泌途径蛋白酶[57]Table 1.2 Proteases of Pichia pastoris vacuole and secretory pathway[57]Enzyme Type Gene Zymogen formVacuole PrA Aspartic PEP4 YesPrB Serine PRB1 YesCpY Serine PRC1 YesCpS Metallo-( Zn2+) CPS1 UnknownApI Metallo-( Zn2+) LAP4 YesApCo Metallo-(Co2+) DAP2 NoDPAP-B Serine SEC11 UnknownSecretory pathway signal peptidase Kex2protease Serine KEX2 YesKex1 carboxypeptidase Serine KEX1 NoDPAP-A Serine STE13 Unknown,predict noYeast aspartyl protease ш Aspartic YAP3 Unknown,predict yes酵母液泡位于基质中，一方面是维持胞内pH和盐离子平衡，储藏盐离子的功能；另一方面，由于液泡中含有大量的非特异性的水解酶，较宽底物范围的内生和异源蛋白酶，液泡是降解蛋白甚至细胞器的一个重要场所，大约80％的蛋白在液泡中降解[57]。
毕赤酵母表达体系中重组蛋白的分离纯化
巴 斯 德 毕 赤 酵 母 （Pichia pastoris）是 在 酿 酒 酵 母 表达体系的基础上， 用其他的酵母菌株构建的、可 高效稳定表达外源基因的新表达系统，即甲醇营养
型 酵 母 （Methy-lotrophicyeast）表 达 系 统 [1]。 作 为 第 2 代酵母表达系统，它不仅克服了大肠杆菌表达系统 不能表达结构复杂的蛋白质、表达的蛋白不能分泌 到细胞外、背景蛋白多、表达水平低等缺点，并且弥 补了哺乳类细胞、 昆虫细胞表达系统操作复杂、表 达水平低、产业化生产造价昂贵的不足，此外，还具 有 其 他 酵 母 表 达 系 统 无 法 比 拟 的 优 越 之 处[2]。
亲 和 层 析 （Affinity Chromatography，AFC）是 利 用
2009 年第 3 期
高炳淼等：毕赤酵母表达体系中重组蛋白的分离纯化
35
偶联亲和配基的亲和吸附介质为固定相亲和吸附 目标产物，使目标产物得到分离纯化的方法。 它具 有很高的选择和分离性能以及较大的载量，只需要 一步处理即可使某待分离的生物大分子从复杂的 混合物中分离出来，达到千倍以上的纯化，并保持 较 高 的 活 性[15]，是 分 离 纯 化 以 及 分 析 生 物 大 分 子 尤 其 是 蛋 白 质 的 有 力 工 具 。 自 从 1967 年 Axen 等[16]用 溴化氰活化多糖凝胶偶联肽和蛋白质的方法成功 制 备 了 固 定 化 酶 ，此 后 亲 和 层 析 在 20 世 纪 70 年 代 有了迅速的发展，目前已得到广泛应用。
2 主要纯化的方法
重组蛋白的分离纯化与传统方式相似，也是利 用其物理和化学性质的差异，即分子的大小、形状、 溶解度、等电点、亲疏水性以及与其它分子的亲和 性 等 性 质 建 立 起 来 的 （表 1）[7]。 从 表 1 可 看 出 ，高 纯 度的蛋白质主要是依靠色谱法技术得到的。 由于重 组蛋白在组织和细胞中仍以复杂混合物的形式存 在，因此到目前为止还没有一个单独或一整套现成 的方法把任何一种蛋白质从复杂的混合物中分离 出来，而只能依据目标蛋白的物理化学性质摸索一 套综合的分离程序，以获得较高纯度的蛋白产品。
毕赤酵母表达手册(详细)
毕赤酵母表达（pichia pastoris expression ）实验手册2010-07-15 10:54:56| 分类：毕赤酵母| 标签：|字号大中小订阅一．毕赤酵母表达常用溶液及缓冲液的配制二．毕赤酵母表达的培养基配制三．主要试验环节的操作 3.1 酵母菌株的分离纯化 3.2 pPICZαA原核宿主菌TOP10F’的活化培养 3.3毕赤酵母表达的试验方法 3.4 毕赤酵母电转化方法 3.5 Pichia酵母表达直接PCR鉴定重组子的方法 3.6 毕赤酵母基因组提取方法 3.7 Mut+表型重组酵母的诱导表达实验关键词：酵母实验毕赤酵母表达 pichia pastoris expression 毕赤酵母酵母菌株大肠杆菌表达系统最突出的优点是工艺简单、产量高、周期短、生产成本低。
然而，许多蛋白质在翻译后，需经过翻译后的修饰加工，如磷酸化、糖基化、酰胺化及蛋白酶水解等过程才能转化成活性形式。
大肠杆菌缺少上述加工机制，不适合用于表达结构复杂的蛋白质。
另外，蛋白质的活性还依赖于形成正确的二硫键并折叠成高级结构，在大肠杆菌中表达的蛋白质往往不能进行正确的折叠，是以包含体状态存在。
包含体的形成虽然简化了产物的纯化，但不利于产物的活性，为了得到有活性的蛋白，就需要进行变性溶解及复性等操作，这一过程比较繁琐，同时增加了成本。
大肠杆菌是用得最多、研究最成熟的基因工程表达系统，当前已商业化的基因工程产品大多是通过大肠杆菌表达的，其主要优点是成本低、产量高、易于操作。
但大肠杆菌是原核生物，不具有真核生物的基因表达调控机制和蛋白质的加工修饰能力，其产物往住形成没有活性的包涵体，需要经过变性、复性等处理，才能应用。
近年来，以酵母作为工程菌表达外源蛋白日益引起重视，原因是与大肠杆菌相比，酵母是低等真核生物，除了具有细胞生长快，易于培养，遗传操作简单等原核生物的特点外，又具有真核生物时表达的蛋白质进行正确加工，修饰，合理的空间折叠等功能，非常有利于真核基因的表达，能有效克服大肠杆菌系统缺乏蛋白翻译后加工、修饰的不足。
巴斯德毕赤酵母表达系统研究进展
巴斯德毕赤酵母表达系统研究进展作者：方园园来源：《绿色大世界》2009年第12期摘要:经过近20年的不断开发和完善,巴斯德毕赤酵母(Pichia pastoris)已经成为目前最成功的真核表达系统之一,被广泛用于医药生产、饲料添加剂开发和科学研究。
介绍了毕赤酵母的生物学特性、常用菌株和表达载体的特点及其研究进展,并阐述了其在外源蛋白的表达方面具有的独特优势。
关键词:毕赤酵母;表达载体;外源蛋白中图分类号:Q78文献标识码:A文章编号:1005-569X(2009)12-0037-031 引言巴斯德毕赤酵母(P.pastoris)是一类在缺乏葡萄糖或甘油时,能利用甲醇做为唯一碳源和能源的酵母菌,具有旺盛的生命力,可以在廉价的非选择性培养基中生长,有较宽的生长pH适应范围(3.0～8.0),有较好的发酵基础,非常有利于实现高密度发酵培养,菌体密度可高达100g干细胞/L,它们生长的适宜温度一般为28～30℃,是常用的外源蛋白表达系统。
2 巴斯德毕赤酵母宿主菌株根据对甲醇利用的情况,P.pastoris可划分为三种表型:第一型,即Mut+型,此型毕赤酵母具有完整的AOX1和AOX2基因,在含甲醇的培养基中生长速率与野生型类似,称为甲醇利用正表型。
绝大多数毕赤酵母为Mut+表型,如GS115和SMD1168;第二型,即MutS型,此型毕赤酵母的AOX1基因部分敲除,被酿酒酵母ARG4基因所取代,AOX2虽然与AOX1有97 %的同源性,但在含甲醇的培养基内该型毕赤酵母生长缓慢,称为甲醇利用慢表型,如KM71(his4 arg4 aox1::ARG4);第三型,即Mut-型,此型毕赤酵母AOX1及AOX2基因均被敲除,细胞不能进行甲醇代谢,无法在甲醇中生长,为甲醇利用负表型,如MC100-3(his4 arg4 aox1::ARG4 aox2::Phis4)。
后两者表达外源蛋白有时优于野生株,且需甲醇较少,有时其表达量甚至高于Mut+型。
毕赤酵母菌
——毕赤酵母
（Pichia.pastoris）
by：文慧霖、裴颖
· 毕赤酵母的介绍 · 毕赤酵母的形态特征 · 毕赤酵母的生产过程 · 毕赤酵母的优缺点 · 毕赤酵母的开发利用
毕赤酵母菌
——全称巴斯德毕赤酵母，是甲醇营养 型酵母中的一类能够利用甲醇作为唯一 碳源和能源的酵母菌。与其它酵母一样， 在无性生长期主要以单倍体形式存在， 当环境营养限制时，常诱导2个生理类 型不同的接合型单倍体 细胞交配，融 合成双倍体。
毕赤酵母结构图
毕赤酵母显微图
优缺点
Pichia.pastoris酵母菌体内无 天然质粒，所以表达载体需与宿 主染色体发生同源重组，将外源 基因表达框架整合于染色体中以 实现外源基因的表达。包括启动 子、外源基因克隆位点、终止序 列、筛选标记等。表达载体都是 穿梭质粒，先在大肠杆菌复制扩 增，然后被导入宿主酵母细胞。 为使产物分泌胞外，表达载体还 需带有信号肽序列。
生产过程
（1）细胞增殖繁衍；
（2）分批流加的过渡阶段（甘油或葡萄糖）； （3）诱导表达阶段（甲醇）；
各阶段碳源都为限制 性基质,其补加速率的 动力学模型是高效表 达的基础。
甲型优点
(1)具有醇氧化酶AOX1基因启动子，这是目前最强，调
控机理最严格的启动子之一；
(2)表达效率高,其表达的外源蛋白可占总表达蛋白的 90%以上,有利于目的蛋白的分离纯化； (3)在简单合成培养基中可实现高密度培养； (4)表达质粒能在基因组的特定位点以单拷贝或多拷贝 的形式稳定整合； (5)由于该酵母可以以甲醇为唯一碳源和能源,而绝大多 数微生物并不能以甲醇为碳源,可以减少污染。Fra bibliotek甲型不足
(1)发酵周期长；
(2)甲醇易燃易爆有毒，存在一 定的危险性； (3)筛选高产菌株需用的药物价 格比较昂贵； (4)培养基和培养条件不成熟。
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营养要求简单，生长快速，适合高密度大规模培养， 很少产生有毒物质，毒性比细菌小，用甲醇不易染菌， 可以减少污染。
高的调控功能，可用于外源基因的表达调控。甲 醇营养型酵母表达系统以巴斯德毕赤酵母(Pichia pastoris)表达系统最为常用。
（3）裂殖酵母(Schizogenesis pombe) 表达系统
只能以分裂和产孢子的方式繁殖的一类酵母， 因此定名为裂殖酵母。与前面几种酵母相比， 它具有更多的与高等真核生物相似的特性： 线 粒体结构、 启动子结构、 转录机制和对蛋白2端 酰基化功能均更接近于哺乳类细胞， 因而正逐 渐成为研究真核细胞分子生物学的模式生物， 它作为外源基因表达系统也开始受到人们的关注。 目前， 已经有多种蛋白利用此系统进行了表达， 如人蛋白凝血因子)G、 细胞色素 V6:8、 人白细 胞介素 YL’D等。此系统表达的外源蛋白更接近 于它们的天然形式 。
的真核生物， 其全序列的测定已于 2019年完成 。 酿酒酵母难于高密度培养，分泌效率低，几乎不分泌分
子量大于30 kD 的外源蛋白质，也不能使所表达的外源 蛋白质正确糖基化，而且表达蛋白质的C 端往往被截短。 因此，一般不用酿酒酵母做重组蛋白质表达的宿主菌。 酿酒酵母本身含有质粒，其表达载体可以有自主复制型 和整合型两种。值得注意的是，酿酒酵母表达的外源蛋 白质往往被高度糖基化，糖链上可以带有40 个以上的 甘露糖残基，糖蛋白的核心寡聚糖链仅含有末端1, 3 甘 露糖，产物的抗原性明显增强。所以，酿酒酵母常常用 来制备亚单位疫苗(如默克乙肝疫苗、口蹄疫疫苗等)。
1993年，Philip Petroleum公司将毕赤酵母表达系统的专利卖给 Research Corporation Technologies公司，并委托Invitrogea公司 进行有关产品销售。
目前毕赤酵母已成功应用于400多种外源蛋白的表达。
酵母作为外源基因表达系统与其 他系统的比较
1、大肠杆菌系统 具有产量高、成本低、生产周期短的特 点，但胞内高水平表达的外源蛋白大多 以不溶解、无活性的包涵体形式存在， 而目前由包涵体中提取高水平、高比活 的目标蛋白的最大问题是回收率极低， 大部分目标蛋白在复性过程中析出丢失。
巴斯德毕赤酵母是甲醇营养型酵母中的一类能够利 用甲醇作为唯一碳源和能源的酵母菌。
酵母菌的形态
毕赤酵母发酵培养形态
毕赤酵母-概述
它是在无性生长期主要以单倍体形式存在，当环境营养限制 时，常诱导2个生理类型不同的接合型单倍体细胞交配，融合 成双倍体。
巴斯德毕赤酵母的另一个生物学特点是，甲醇代谢所需的醇 氧化酶被分选到过氧化物酶体中，形成区域化。以葡萄糖作 碳源时，菌体中只有1个或很少几个小的过氧化物酶体，而以 甲醇作碳源时，过氧化物酶体几乎占到整个细胞体积80%， AOX增至细胞总蛋白的35%-40%。因此，当在AOX基因前利 用同源重组方式插入外源蛋白基因时，可获得大量表达。同 时，根据甲醇酵母这种可以形成过氧化物酶体的特性，既可 利用该系统表达一些毒性蛋白和易被降解的酶类，也可用以 研究细胞特异区域化的生物发生及其机制和功能，为高等动 物类似的研究提供启示
2、杆状病毒系统
采用先瞬时感染，然后裂解感染昆虫细 胞的方式产生重组蛋白，此系统表达的 蛋白折叠正确且被糖基化修饰，但有时 糖基化序列与天然的不同，且蛋白产量 不稳定。
3、哺乳动物细胞表达系统
生产的蛋白具有天然构象，稳定性好， 糖基化模式没有发生改变，但哺乳动物 细胞培养成本高，条件要求严格，蛋白 表达水平较低。
4、酵母表达系统
酵母是低等真核生物，具有细胞生长快， 易于培养，遗传操作简单等原核生物的 特点，又具有真核生物对表达的蛋白质 进行正确加工，修饰，合理的空间折叠 等功能，非常有利于真核基因的表达， 能有效克服大肠杆菌系统缺乏蛋白翻译 后加工、修饰等不足.
几种常用酵母表达系统
（1）酿酒酵母(Saccharomyces cerevisiae)表达系统 作为真核生物的模式菌， 酿酒酵母是目前了解最完全
八十年代，Philips Petroleum Company与Salk Institute Biotechnology/Industrial Associates, Inc.(SIBIA, La Jolla, CA)开 始联合开发毕赤酵母系统。1985年SIBIA公司的研究人员Ellis 等首次成功分离了甲醇氧化酶基因及其启动子。同年，Cregg 等报道了巴斯德毕赤酵母作为宿主成功表达外源蛋白。
毕赤酵母开发利用历史
1969年，koichi Ogata首次报道了某些酵母菌能够利用甲醇作为 唯一碳源和能源，七十年代美国 Philips Petroleum Company成 功开发了廉价的培养基和高细胞密度方法培养甲醇营养型巴斯 德毕赤酵母（Pichia pastoris）生产单细胞蛋白作为饲料。
该表达系统质粒易丢失， 传代不稳定。
（2）甲醇营养型酵母表达系统 甲醇营养型酵母包括汉逊酵母属(Hansenula)，毕 赤酵母属(Pichia)，球拟酵母属(Torulopsis)等，能
在以甲醇为唯一能源和碳源的培养基上生长，甲
醇可以诱导它们表达甲醇代谢所需的酶，如醇氧 化酶I(AOX1)， 二羟丙酮合成酶(DHAS)、甲酸 脱氢酶(FMD)等。 AOX1的甲醇诱导表达量可占 胞内总蛋白质的20％-30％，表明AOX1 的合成 受转录水平的调控。AOX1 启动子(PAox )具有较
酿酒酵母优缺点
优点
不产生毒素，遗传背景清楚，安全性好， 易进行 操作
酿酒酵母是真核生物， 可以对蛋白进行翻译后加 工。
表达产物可分泌表达， 易于纯化。
酿酒酵母生长迅速， 工艺简单， 成本低。
缺点
对真核基因产物的翻译后加工与高等真核生物有 所不同， 重组蛋白常发生超糖基化。
酿酒酵母不易进行高密度发酵， 表达产物产量低。 分泌效率低， >30kD的蛋白质几乎不分泌。