巴斯德毕赤酵母(Pichia pastoris)表达系统综述

毕赤酵母表达知识归纳1a.配制500×BIOTIN stock solution（0.02％）有这么3种方案：1、懒人是将Biotin直接溶在去离子水中，放过夜，基本就能溶；2、急性子是将溶液配成0.02N的NaOH，就很容易溶解了；3、水浴加热，温度不能高于50度。

D－生物素是具有生物活性的生物素，也就是vitaminH。

在毕赤酵母代谢过程中，作为多种酶的辅基起作用。

天然培养基中一般可以不单独添加，因为YNB中、酵母粉、蛋白胨中均含有一定量的生物素，但是做高密度发酵还是必须要添加的。

b.有几个比较迷惑的问题请教大家：(很典型的小问题）1、制感受态细胞，OD多少比较好？pyrimidine 战友的方法：取1mlGS115过夜培养物(OD约6-10) 分装到1.5ml EP管中。

说明书还有一些文献是说在1.3左右效率高，再高了效率会很低2、关于高效转化法，文献说用(LiAc)，而invitrogen的说明书说转化毕赤酵母用(LiAc)没用，要用LiCl。

Lithium acetate does not work with Pichia pastoris. Use only lithium chloride.3、YNB到底能高温灭么？有的说能有的说不能。

过滤灭菌的怎么操作？我是把滤器装好膜绑到瓶口用纱布盖上，报纸包上，瓶盖放烧杯里单灭。

然后把配好的溶液用注射器一点点推进去。

4、葡萄糖为什么在YPD里一起灭颜色很深，单灭则不会。

该115度还是121度灭？网上搜了下，都有人用！5、电转化参数用400欧还是200欧？有的用400，有的还专门说不是用400。

都是从园里看到的！电击参数：1.5KV，25uF，200欧姆（不是400）6、电转后，在MD平板上长的应该就是整合了目的基因的重组子了吧？如果不想筛高拷贝的，是否PCR验证一下即可？网友的回答：ynb最好不灭菌，我是0.22um过滤处理的。

invitrogen手册上可以灭菌的。

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。

毕赤酵母表黑系统之阳早格格创做Mut+战Muts毕赤酵母中有二个基果编码醇氧化酶——AOX1及AOX2，细胞中大普遍的醇氧化酶是AOX1基果产品，甲醇可稀切安排、诱导AOX1基果的下火仄表黑，较典型的是占可溶性蛋黑的30%以上.AOX1基果调控分二步：压制/去压制体制加诱导体制.简朴去道，正在含葡萄糖的培植基中，纵然加进诱导物甲醇转录仍受压制.为此，用甲醇举止劣化诱导时，推荐正在苦油培植基中培植.注意纵然正在苦油中死少(去压制)时，仍缺乏以使AOX1基果达到最矮火仄的表黑，诱导物甲醇是AOX1基果可辨表黑火仄所必须的.AOX1基果已被分散，含AOX1开用子的量粒可用去促进编码中源蛋黑的手段基果的表黑.AOX2基果与AOX1基果有97%的共源性，然而正在甲醇中戴AOX2基果的菌株比戴AOX1基果菌株缓得多，通过那种甲醇利用缓缓表型可分散Muts菌株.正在YPD(酵母膏、蛋黑胨、葡萄糖)培植基中，不管是Mut+仍旧Muts其正在对付数期删殖一倍的时间约莫为2h.Mut+战Muts菌株正在不甲醇存留的情况下死少速率是一般的，存留甲醇的情况下，Mut+正在对付数期删殖一倍的时间约莫为4至6个小时，Muts正在对付数期删殖一倍的时间约莫为18个小时.菌株GS115、X-33、KM71战SMD1168的辨别GS115、KM71战SMD1168等是用于表黑中源蛋黑的毕赤酵母受体菌，与酿酒酵母相比，毕赤酵母不会使蛋黑过糖基化，糖基化后有好处蛋黑的溶解或者产死粗确的合叠结构.GS115、KM71、SMD1168正在组氨酸脱氢酶位面(His4)有突变，是组氨酸缺陷型，如果表黑载体上携戴有组氨酸基果，可补偿宿主菌的组氨酸缺陷，果此不妨正在不含组氨酸的培植基上筛选变化子.那些受体菌自收突形成组氨酸家死型的概率普遍矮于10-8.GS115表型为Mut+，沉组表黑载体变化GS115后，少出的变化子大概是Mut+，也大概是Muts(载体与代AXO1基果)，不妨正在MM战MD 培植基上审定表型.SMD1168战GS115类似，然而SMD1168基果组中的Pep4基果爆收突变，是蛋黑酶缺陷型，可落矮蛋黑酶对付中源蛋黑的落解效率.其中X-33由于是家死型，果此耐受性比较佳，如果担心变化率的话不妨思量那种酵母菌，而X33与GS115一般皆是属于MUT＋表示型，也便是道不妨正在含甲醇的培植基中赶快死少，然而是传闻会对付中源基果表黑灵验率，KM71的亲原菌正在粗氨酸琥珀酸裂解酶基果(arg4)有突变，正在不含粗氨酸的培植基中不克不迭死少.用家死型ARG4基果(约2kb)拔出到克隆的家死型AOX1基果的BamHI(AOX1基果15/16暗号子)及SalI(AOX1基果227/228暗号子)位面，与代了AOX1基果16-227暗号子，此结构变化至KM71亲原菌(arg4his4)中，分散爆收KM71 MutsArg+His-菌株，Arg+变化子遗传领会隐现家死型AOX1被aox1::ARG4结构所与代，所以KM71所有变化子皆是Muts表型.AOX1位面不被真足缺得，表面上可用您的手段结构通过基果与代要领替换aox1::ARG4结构，那样沉组菌株的表型是His+MutsArg-，那表示着沉组菌株死万古需粗氨酸.然而仅增加粗氨酸本去不克不迭真足缓战arg4突变的效率，arg4菌株正在含粗氨酸的最小培植基中不克不迭很佳天死少.果此不推荐正在KM71中通过与代aox1::ARG4结构去赢得His＋变化子.普遍去道，如果是胞内表黑，应尽管用Muts细胞，那样得到的蛋黑产品中醇氧化酶蛋黑量较少而手段蛋黑量相对付较多，使下游杂化更易举止.而对付于分泌蛋黑的表黑，无论是甲醇利用缓(Muts)仍旧甲醇利用快(Mut+)的细胞皆可应用.基果沉组Pichia.pastoris酵母菌体内无天然量粒，所以表黑载体需与宿主染色体爆收共源沉组，将中源基果表黑框架调整于染色体中以真止中源基果的表黑，包罗开用子、中源基果克隆位面、末止序列、筛选标记表记标帜等.细菌内共源沉组被认为是沉组量粒构修历程的易面，果为已线性化的环状量粒之间爆收共源沉组的几率非常矮，所以沉组变化载体必须用特定的节制性内切酶举止线性化处理.那种处理的手段是预防随机拔出沉组时量粒正在功能区断开，制成手段基果表黑得活，让共源沉组以指定的办法爆收.表黑载体主要分为以下几类：(1)胞内表黑载体主要有pHIL-D2、pA0815、pPIC3K、pPICZ、pHWO10，pGAPZ、pGAPZa(Invitrogen)等.该类载体不妨将手段基果表黑正在胞内，不妨预防毕赤酵母的糖基化，主要符合于那些不克不迭被糖基化相闭基果的表黑；(2)分泌型表黑载体主要有pPIC9、pHIL-S1、pPICZα、pYAM75P等.由于毕赤酵母自己的泌内源蛋黑非常少，将中源蛋黑分泌到胞中，非常有好处手段蛋黑量的杂化及聚集.时常使用的分泌的旗号序列主假如由89个氨基酸组成的α接配果子(α-factor)的带领；(3)多拷贝拔出表黑载体如pPIC9K，pPIC3.5K.正在某些情况下，毕赤酵母中沉组基果多拷贝调整可减少所需蛋黑的表黑量.该载体均可用于正在体内(pPIC3.5K, pPIC9K)或者体中(pAO815)爆收并分散多拷贝拔出，共时可检测减少沉组基果的拷贝数是可减少蛋黑表黑量.体内调整可通过下遗传霉素抗性筛选大概的多拷贝拔出，而体中调整可通过对接爆收中源基果的串联拔出.正在GS115中筛选His+Mut+变化子：用SalI或者StuI线性化量粒变化GS115后，大多正在His4位面上爆收沉组，大普遍变化子是Mut+表型；然而由于量粒含有AOX1基果序列，有大概正在AOX1位面爆收沉组，损害家死型AOX1基果，爆收His+Muts变化子，则需要正在MD及MM仄板上检测可证据His+ Mut+变化子.毕赤酵母表黑时常使用培植基10×YNB(13.4%的无氨基酸酵母氮源)，134gYNB固体溶于1L蒸馏火，过滤灭菌，4℃保存.YPD真足培植基：酵母提与物10 g/L，蛋黑胨20 g/L，葡萄糖20 g/L(固体培植基含1.5%琼脂).变化培植基RDB：每100mL加进山梨醇18g(186 g/L)，琼脂糖2g(20g/L)121℃灭菌20分钟，而后待温度落至60℃以去正在超洁台上加进10×YNB 10mL(13.4 g/L)，10×葡萄糖10mL(20 g/L)，500×死物素0.2mL(4×10-4g/L)，100×AA 1mL.混匀，倒仄板(灭菌时只加进80ml火即可).采用培植基MD(最小葡萄糖)：配100mL，背80mL火中加进琼脂糖2g(20 g/L)121℃灭菌20分钟，待温度落至60℃以去正在超洁台上加进10×YNB10mL(13.4 g/L)，10×葡萄糖10mL(20 g/L)，500×死物素0.2mL(4×10-4g/L).采用培植基MM(最小甲醇)：配100mL，背90mL火中加进琼脂糖2g(20 g/L) 121℃灭菌20分钟，待温度落至60℃以去正在超洁台上加进10×YNB 10mL(13.4 g/L)，500×死物素0.2mL(4×10-4g/L)，0.5mL甲醇(0.5%).诱导表黑培植基BMGY：配1L，酵母提与物10 g/L，蛋黑胨20 g/L，3g/L K2HPO4，11.8g/L KH2PO4，加火至890mL，121℃灭菌20分钟，而后待温度落至60℃以去正在超洁台上加进10×YNB 100mL(13.4 g/L)，500×死物素1mL(4×10-4g/L)，苦油10mL.诱导表黑培植基BMMY：酵母提与物10g/L，蛋黑胨20 g/L，3g/LK2HPO4，11.8g/L KH2PO4，加火至895mL，121℃灭菌20分钟，而后待温度落至60℃以去正在超洁台上加进100×YNB 100mL(13.4 g/L)，500×死物素1mL(4×10-4g/L)，甲醇5mL.BMGY/BMMY含酵母浸出物及蛋黑胨，可宁静分泌蛋黑，遏止或者缩小分泌蛋黑的领会.如果手段蛋黑对付中性PH蛋黑酶敏感的话，可正在无缓冲培植基(MGY、MM)中表黑.如果不凭证道明您的分泌蛋黑对付中性PH 值蛋黑酶敏感，修议开初表黑时用BMMY.如果表黑蛋黑落解了，测验考查正在无缓冲培植基中举止表黑.如果以上条件仍不克不迭灵验预防蛋黑落解，可将基果转进SMD1168中，该菌株表型是his4pep4，缺得了蛋黑酶，变化与表黑步调与GS115相共，也可用于大规模收酵.用考马斯明蓝G-250测蛋黑含量。

Pichia酵母表达系统使用心得摘要：Pichia酵母表达系统广泛应用于外源基因表达。

生物通编者按：甲醇酵母表达系统有不少优点，其中以Invitrogen公司的Pichia酵母表达系统最为人熟知，并广泛应用于外源蛋白的表达。

虽然说酵母表达操作简单表达量高，但是在实际操作中，并不是每个外源基因都能顺利得到高表达的。

不少人在操作中会遇到这样那样的问题，生物通编者特地收集了部分用户在使用EasySelect Pichia Expression System这个被誉为最简单的毕赤酵母表达的经典试剂盒过程中的心得体会。

其中Xiang Yang是来自美国乔治城大学（Georgetown University）Lombardi癌症中心（Lombardi Cancer Center），部分用户来自国内。

+ 表示优胜于；- 表示不如；= 表示差不多EasySelect Pichia Expression System产品性能：优点——使用简单，表达量高，His-tag便于纯化缺点——酵母表达蛋白有时会出现蛋白切割问题全面产品报告及心得体会：巴斯德毕赤酵母（Pichia pastoris）是一种能高效表达重组蛋白的酵母品种，一方面由于其是属于真核生物，因此表达出来的蛋白可以进行糖基化修饰，另一方面毕赤酵母生长速度快，可以将表达的蛋白分泌到培养基中，方便蛋白纯化。

毕赤酵母表达载体pPICZ在多克隆位点（MCR）3'端带有his-tag和c-myc epitopes，这些tag有利于常规检测和纯化，而且在MCR5'端引入了alpha factor（α-factor）用以增加表达，并且在表达后α-factor可以自动被切除。

在进行克隆的时候，如果你选择的是EcoRI，那么只需在目标蛋白中增加两个氨基酸序列即可完成。

另外pPICZ系列选用的是Zeocin抗生素作为筛选标记，而诱导表达的载体需要甲醇——甲醇比一般用于大肠杆菌表达诱导使用的IPTG便宜。

毕赤酵母表达系统毕赤酵母表达系统前言：所用表达质粒有pPIC3.5K,pAO815用于胞内表达，而pPIC9K用于分泌表达，所有载体均利用AOX1启动子来诱导高水平表达。

抗性选择：最有效的筛选遗传霉素抗性及高抗性克隆的程序需要先对HIS＋转化子进行选择，再进行不同水平遗传霉素抗性筛选。

毕赤菌株表型：毕赤酵母菌GS115 及KM71 在组氨酸脱氢酶位点（His4）有突变,因而不能合成组氨酸，所有表达质粒都有HIS4 基因可与宿主进行互补，通过不含组氨酸的培养基来选择转化子。

GS115 及KM71都可在复合培养基如YPD（YEPD）及含组氨酸的最小培养基中生长。

转化之前，GS115 及KM71 都不能在最小培养基中生长，因为它们是His-。

培养温度：毕赤酵母生长温度为28-30度（液体、平板、斜面）。

在32 度以上诱导生长时，对蛋白表达有害，甚至会导致细胞死亡。

贮存：贮存细胞几周或几月，用YPD培养基或YPD 琼脂斜面1 挑取所需菌株单克隆在YPD 平板上划线生长；2 挑取单克隆转移至YPD进行穿刺培养，30 度2 天；3 细胞在4 度可放几周几月或几年，存于－80度1 挑取所需菌株单克隆在YPD 中过夜培养；2 收集细胞，在含15%甘油的YPD 中悬浮至终OD600 为50-100（大约2.5-5.0×109细胞/ml）；3 细胞先用液氮或干冰/酒精浴中冰冻再贮存于-80 度。

注意：在4 度或－80 度长期保存后，用之前建议在MM、MD 或MGY 平板上划线培养以检测His+转化子的表型是否正确及其活力。

以质粒pPIC9K，酵母Pichia pastoris GS115为例说明做法。

载体pPIC9K酶切为点线性化质粒DNA：建议使用下列方法线性化载体以获得Mut+及Muts重组子，可能其中一个会比另一个更利于表达多拷贝重组子。

如果只想得到Muts 重组子，使用KM71 菌株。

单个十字交换事件可比双重十字交换更容易、更有效地获得Muts 重组菌（例如：插入AOX1或his4 而不是取代AOX1）。

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。