Man5GlcNAc2哺乳动物甘露糖型糖蛋白的毕赤酵母表达系统构建
毕赤酵母表达手册
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毕赤酵母多拷贝表达载体试剂盒用于在含多拷贝基因的毕赤酵母菌中表达并分离重组蛋白综述：基本特征：作为真核生物，毕赤酵母具有高等真核表达系统的许多优点：如蛋白加工、折叠、翻译后修饰等。
不仅如此，操作时与E.coli及酿酒酵母同样简单。
它比杆状病毒或哺乳动物组织培养等其它真核表达系统更快捷、简单、廉价，且表达水平更高。
同为酵母，毕赤酵母具有与酿酒酵母相似的分子及遗传操作优点，且它的外源蛋白表达水平是后者的十倍以至百倍。
这些使得毕赤酵母成为非常有用的蛋白表达系统。
与酿酒酵母相似技术：许多技术可以通用：互补转化基因置换基因破坏另外，在酿酒酵母中应用的术语也可用于毕赤酵母。
例如：HIS4基因都编码组氨酸脱氢酶；两者中基因产物有交叉互补；酿酒酵母中的一些野生型基因与毕赤酵母中的突变基因相互补，如HIS4、LEU2、ARG4、TR11、URA3等基因在毕赤酵母中都有各自相互补的突变基因。
毕赤酵母是甲醇营养型酵母：毕赤酵母是甲醇营养型酵母，可利用甲醇作为其唯一碳源。
甲醇代谢的第一步是：醇氧化酶利用氧分子将甲醇氧化为甲醛，还有过氧化氢。
为避免过氧化氢的毒性，甲醛代谢主要在一个特殊的细胞器－过氧化物酶体－里进行，使得有毒的副产物远离细胞其余组分。
毕赤酵母表达系统简介
巴斯德毕赤酵母及启动子1.1 毕赤酵母表达系统简介随着蛋白异源表达的飞速发展，越来越多的表达系统被建立并得到应用。
酵母作为单细胞真核生物，因具有比较完备的基因表达调控机制和对表达产物的加工修饰能力，仍表现出不可比拟的优势。
以甲醇营养型酵母（Methylotrophic yeast）-毕赤酵母为代表的第二代酵母表达系统，是近年来被公认的最有效的外源蛋白表达系统之一，已有多种外源蛋白在该宿主系统中获得了成功表达[1]。
作为生产外源蛋白的重要宿主菌，依靠其各种不同功能的表达载体，已经得到广泛的应用。
表达的蛋白质包括酶、膜蛋白、抗原、抗体和调节蛋白等[2,3]。
毕赤酵母（Pichia pastoris）表达系统是近年来发展迅速、应用广泛的一种真表达系统。
它是甲醇营养型酵母菌，有两个乙醇氧化酶（alcohol oxidase，Aox）码基因AOX1和AOX2，两者序列相似，AOX1基因严格受甲醇诱导和调控。
当甲醇为唯一碳源时，AOX1启动子可被甲醇诱导，启动乙醇氧化酶的表达，从而用甲醇进行代谢[4]。
含AOX1启动子的质粒可用来促进编码外源蛋白的目的因的表达。
随着Invitrogen公司开发的一系列毕赤酵母表达试剂盒的应用，目前用该统已成功表达出了数以千计的来自细菌、真菌、原生动物、植物、无脊椎动物、包括人在内的脊椎动物以及病毒等的具有生物学功能的外源蛋白或蛋白结构[5,6]。
1.1.1 P.Pastoris表达载体及其元件由于毕赤酵母没有稳定的附加质粒，表达载体需与宿主染色体发生同源重组，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整合表达的优点在于保持外源基因稳定性并可产生多拷贝基因。
典型的毕赤氏酵母表达载体含有醇氧化酶基因的调控序列，主要的结构包括：5′AOX1启动子片段、多克隆位点(MCS)、转录终止和polyA形成基因序列(TT)、筛选标记(His4或Zeocin)、3′AOX1基因片段，作为一个能在大肠杆菌中繁殖扩增的穿梭质粒，它还有部分pBR322质粒或COLE1序列。
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。
β-甘露聚糖酶基因和木聚糖酶基因在毕赤母中的共表达
β-甘露聚糖酶基因和木聚糖酶基因在毕赤母中的共表达李剑芳;赵顺阁;邬敏辰;张慧敏;魏喜换【期刊名称】《食品与生物技术学报》【年(卷),期】2012(031)011【摘要】为实现β-甘露聚糖酶基因和木聚糖酶基因在毕赤酵母中的共表达,作者将经SalⅠ线性化的pPIC9K-xynⅡ电转化至工程菌GS115/Anman5A中,经G418浓度梯度筛选后获得能同时高产β-甘露聚糖酶和木聚糖酶的双重重组子GS115/Anman5A-xynⅡ.SDS-PAGE显示目的蛋白的相对分子质量分别约为52 000,24 100.摇瓶发酵筛选出产酶活性最强的转化株,命名为GSM X2,随后对该菌株的表达条件进行初步优化,优化后的表达条件为:诱导时间120 h,培养基起始pH 值为7.0,甘油添加量为1.5％,甲醇添加量为1.5％.在此培养条件下,产β-甘露聚糖酶和木聚糖酶的活性分别达到37.1 U/mL和193.6 U/mL.【总页数】6页(P1136-1141)【作者】李剑芳;赵顺阁;邬敏辰;张慧敏;魏喜换【作者单位】江南大学食品学院,江苏无锡214122;江南大学食品学院,江苏无锡214122;江南大学医药学院,江苏无锡214122;江南大学食品学院,江苏无锡214122;江南大学食品学院,江苏无锡214122【正文语种】中文【中图分类】S37【相关文献】1.果胶裂解酶、木聚糖酶及果胶裂解酶、木聚糖酶基因在毕赤酵母中的表达 [J], 高秋芳;郭安平;孔华;邓伟科;贺立卡2.黑曲霉糖化酶和木聚糖酶基因在工业用酿酒酵母中的共表达 [J], 李海燕;毛爱军;何永志;董志扬3.乙醇酸氧化酶基因在巴斯德毕赤氏酵母中的表达 [J], 吴旭亚;何冰芳;李霜;刘志斌;欧阳平凯4.深黄被孢霉Δ6-脂肪酸脱氢酶基因在毕赤氏酵母SMD1168中表达的研究 [J], 王长远;全越;沈冰蕾;姚笛;于长青5.羰基还原酶基因与葡萄糖脱氢酶基因在大肠杆菌中的共表达及其在不对称还原产麻黄碱中的初步应用 [J], 宇文伟刚;张梁;王正祥;石贵阳因版权原因，仅展示原文概要，查看原文内容请购买。
毕赤酵母表达外源蛋白糖基化研究进展
毕赤酵母表达外源蛋白糖基化研究进展杨婕【摘要】Pichia pastoris as the host for the expression of recombinant proteins, has not only the advantages of prokaryotic expression system such as inexpensive in culture, ease of genetical manipulation, high levelsof protein expression, but also has post-translational protein processing capabilities like other higher eukaryotes such as protein folding, formation of disulfide bond and glycosylation. Gly-cosylation is an important form of posttranslational modification in secreted proteins and affects the structure and function of these pro-teins. In this review, we discuss protein glycosylation and humanized glycosylation engineering in Pichia pastoris.%毕赤酵母作为表达外源蛋白的宿主，不仅具有原核生物表达系统的优点，如培养成本低、遗传操作简单、表达效率高等，还可以对表达的外源蛋白进行翻译后修饰加工，如蛋白质折叠、二硫键形成和糖基化等，因而有其独特的优势。
在分泌蛋白修饰中，糖基化是一种重要修饰方式，影响蛋白的结构与功能。
毕赤酵母表达系统资料整理
毕赤酵母表达系统之马矢奏春创作Mut+和Muts毕赤酵母中有两个基因编码醇氧化酶——AOX1及AOX2,细胞中年夜大都的醇氧化酶是AOX1基因产物,甲醇可紧密调节、诱导AOX1基因的高水平表达,较典范的是占可溶性卵白的30%以上.AOX1基因调控分两步：抑制/去抑制机制加诱导机制.简单来说,在含葡萄糖的培养基中,即使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,推荐在甘油培养基中培养.注意即使在甘油中生长(去抑制)时,仍缺乏以使AOX1基因到达最低水平的表达,诱导物甲醇是AOX1基因可辨表达水平所必需的.AOX1基因已被分离,含AOX1启动子的质粒可用来增进编码外源卵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基因菌株慢很多,通过这种甲醇利用缓慢表型可分离Muts菌株.在YPD(酵母膏、卵白胨、葡萄糖)培养基中,不论是Mut+还是Muts其在对数期增殖一倍的时间年夜约为2h.Mut+和Muts菌株在没有甲醇存在的情况下生长速率是一样的,存在甲醇的情况下,Mut+在对数期增殖一倍的时间年夜约为4至6个小时,Muts在对数期增殖一倍的时间年夜约为18个小时.菌株GS115、X-33、KM71和SMD1168的区别GS115、KM71和SMD1168等是用于表达外源卵白的毕赤酵母受体菌,与酿酒酵母相比,毕赤酵母不会使卵白过糖基化,糖基化后有利于卵白的溶解或形成正确的折叠结构.GS115、KM71、SMD1168在组氨酸脱氢酶位点(His4)有突变,是组氨酸缺陷型,如果表达载体上携带有组氨酸基因,可赔偿宿主菌的组氨酸缺陷,因此可以在不含组氨酸的培养基上筛选转化子.这些受体菌自发突酿成组氨酸野生型的概率一般低于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培养基上鉴定表型.SMD1168和GS115类似,但SMD1168基因组中的Pep4基因发生突变,是卵白酶缺陷型,可降低卵白酶对外源卵白的降解作用.其中X-33由于是野生型,因此耐受性比力好,如果担忧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＋暗示型,也就是说可以在含甲醇的培养基中快速生长,可是据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)有突变,在不含精氨酸的培养基中不能生长.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+转化子遗传分析显示野生型AOX1被aox1::ARG4结构所取代,所以KM71所有转化子都是Muts表型.AOX1位点没有被完全缺失,理论上可用你的目的结构通过基因取代方法替换aox1::ARG4结构,这样重组菌株的表型是His+MutsArg-,这意味着重组菌株生长时需精氨酸.但仅添加精氨酸其实不能完全缓和arg4突变的影响,arg4菌株在含精氨酸的最小培养基中不能很好地生长.因此不推荐在KM71中通过取代aox1::ARG4结构来获得His ＋转化子.一般来说,如果是胞内表达,应尽量用Muts细胞,这样获得的卵白产物中醇氧化酶卵白量较少而目的卵白量相对较多,使下游纯化更易进行.而对分泌卵白的表达,无论是甲醇利用慢(Muts)还是甲醇利用快(Mut+)的细胞都可应用.基因重组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、筛选标识表记标帜等.细菌内同源重组被认为是重组质粒构建过程的难点,因为未线性化的环状质粒之间发生同源重组的几率非常低,所以重组转移载体必需用特定的限制性内切酶进行线性化处置.这种处置的目的是防止随机拔出重组时质粒在功能区断开,造成目的基因表达失活,让同源重组以指定的方式发生.表达载体主要分为以下几类：(1)胞内表达载体主要有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,主要适合于那些不能被糖基化相关基因的表达；(2)分泌型表达载体主要有pPIC9、pHIL-S1、pPICZα、pYAM75P等.由于毕赤酵母自己的泌内源卵白非常少,将外源卵白分泌到胞外,非常有利于目的卵白质的纯化及积累.经常使用的分泌的信号序列主要是由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.在某些情况下,毕赤酵母中重组基因多拷贝整合可增加所需卵白的表达量.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,同时可检测增加重组基因的拷贝数是否增加卵白表达量.体内整合可通过高遗传霉素抗性筛选可能的多拷贝拔出,而体外整合可通过连接发生外源基因的串连拔出.在GS115中筛选His+Mut+转化子：用SalI或StuI线性化质粒转化GS115后,年夜多在His4位点上发生重组,年夜大都转化子是Mut+表型；然而由于质粒含有AOX1基因序列,有可能在AOX1位点发生重组,破坏野生型AOX1基因,发生His+Muts转化子,则需要在MD及MM平板上检测可证实His+ Mut+转化子.毕赤酵母表达经常使用培养基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完全培养基：酵母提取物10 g/L,卵白胨20 g/L,葡萄糖20 g/L(固体培养基含 1.5%琼脂).转化培养基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℃以后在超净台上加入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).选择培养基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℃以后在超净台上加入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).选择培养基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℃以后在超净台上加入10×YNB 10mL(13.4 g/L),500×生物素0.2mL(4×10-4g/L),0.5mL甲醇(0.5%).诱导表达培养基BMGY：配1L,酵母提取物10 g/L,卵白胨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℃以后在超净台上加入10×YNB 100mL(13.4 g/L),500×生物素1mL(4×10-4g/L),甘油10mL.诱导表达培养基BMMY：酵母提取物10g/L,卵白胨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℃以后在超净台上加入100×YNB 100mL(13.4 g/L),500×生物素1mL(4×10-4g/L),甲醇5mL. BMGY/BMMY含酵母浸出物及卵白胨,可稳定分泌卵白,阻止或减少分泌卵白的分解.如果目的卵白对中性PH卵白酶敏感的话,可在无缓冲培养基(MGY、MM)中表达.如果没有证据证明你的分泌卵白对中性PH值卵白酶敏感,建议开始表达时用BMMY.如果表达卵白降解了,检验考试在无缓冲培养基中进行表达.如果以上条件仍不能有效防止卵白降解,可将基因转入SMD1168中,该菌株表型是his4pep4,缺失了卵白酶,转化与表达法式与GS115相同,也可用于年夜规模发酵.用考马斯亮蓝G-250测卵白含量。
一种强化乳铁蛋白表达的毕赤酵母及其构建方法与应用
一、概述乳铁蛋白是一种存在于哺乳动物乳汁中的重要蛋白质，具有丰富的营养价值和生物活性。
其在免疫调节、抗氧化、抗菌、抗病毒等方面具有重要作用，因此受到了广泛的关注。
而毕赤酵母是一种常见的真菌微生物，可以被用于多种重要蛋白的表达和生产。
构建一种强化乳铁蛋白表达的毕赤酵母具有重要的研究意义和应用价值。
二、毕赤酵母的特点1. 毕赤酵母的生物学特性毕赤酵母（Pichia pastoris）是一种酵母，通过改良的形态和工程学技术，可以高效表达和分泌异源蛋白，其优点包括a. 生长速度快b. 表达异源蛋白能力强c. 分泌蛋白质的能力优秀2. 毕赤酵母在蛋白表达中的应用毕赤酵母在蛋白表达中应用广泛，主要包括医药、工业、生物学等领域。
通过毕赤酵母表达系统，可以实现对多种重要蛋白的高效表达和大规模生产。
三、强化乳铁蛋白表达的毕赤酵母构建方法1. 选择适合的毕赤酵母表达载体通过对不同毕赤酵母表达载体的筛选和比较，选取适合乳铁蛋白表达的载体。
2. 乳铁蛋白基因的克隆和插入通过PCR扩增、酶切、连接等分子生物学技术，得到乳铁蛋白基因的重组表达载体。
3. 转化和筛选将表达载体转化至毕赤酵母菌株中，进行筛选得到高效表达乳铁蛋白的毕赤酵母菌株。
4. 条件优化与表达通过调节培养基、温度、pH值等条件，优化乳铁蛋白的表达条件，提高其表达量和纯度。
四、强化乳铁蛋白表达的毕赤酵母的研究进展1. 表达量和纯度的提高通过使用不同的表达载体、优化表达条件等手段，可以显著提高乳铁蛋白的表达量和纯度，满足不同应用的需求。
2. 抗性因子的引入引入抗性因子可以提高毕赤酵母对特定抗生素的抗性，从而提高其在生产中的稳定性和可操作性。
3. 结构与功能分析通过结构生物学等技术手段对乳铁蛋白在毕赤酵母中的结构和功能进行深入研究，为其应用提供理论基础。
五、强化乳铁蛋白表达的毕赤酵母在食品、医药等领域的应用1. 食品领域强化乳铁蛋白表达的毕赤酵母可以用于生产高营养价值的乳制品，如乳粉、奶酪等，提高其抗菌、抗氧化等功能。
毕赤酵母N_糖基化改造的研究进展
毕赤酵母 N 2糖基化进一步改造需要去除甘露 糖 , 由 M an8 GlcNAc2 结 构 转 变 为 M an5 GlcNA c2 结 构 。α1, 2甘露糖苷酶 Ⅰ (M ns Ⅰ)广泛存在于各种 真核生物的内侧高尔基体 ,主要作用是降解甘露糖 残基 ,M aras等 [ 10 ]将里氏木霉的 M ns Ⅰ基因引入毕 赤酵母得到表达 , Callewaert等 [ 11 ] 将内质网定位序 列 HDEL 引入里氏木霉的 M ns Ⅰ基因在毕赤酵母 中进行表达 ,发现大多数 α1, 2 甘露糖都被切除 ords] P ich ia pastoris; N 2glycosylation; glycop rotein
毕赤酵母 ( P ich ia pastoris)表达系统是 20 世纪 80年代初期发展起来的一种新型的外源蛋白表达 系统 ,它既具有原核表达系统操作简易 、易于培养 、 生长速度快 、表达量高 、成本低等优点 ,还具有真核 生物表达系统的对外源蛋白的翻译后修饰等特点 , 如糖基化 、蛋白磷酸化等 。目前 ,已有越来越多的药 用重组蛋白通过酵母表达系统表达 ,如肿瘤坏死因 子和表皮生长因子等 ,其中有 300 多种外源蛋白在 该表达系统中获得了高效表达 ,而明胶的表达量甚 至达到了 14. 8 g·L - 。 1 [ 1 ]
中国新药杂志 2008年第 17卷第 14期
来进行 N 2糖基化改造 。在敲除 O ch1 基因的基础 上 ,将不同来源的 M ns Ⅰ催化区与不同来源的内质 网 ,高尔基体定位序列融合表达 ,通过高通量筛选 , M aldi2tof法检测报告蛋白 K3的糖链结构 ,得到了能 够高效表达 M an5 GlcNAc2 结构的菌株 。
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Man5GlcNAc2哺乳动物甘露糖型糖蛋白的毕赤酵母表达系统构建杨晓鹏1，刘波1，宋淼1,2，巩新1，唱韶红1，薛奎晶1,2，吴军1【摘要】摘要: 蛋白的糖基化对蛋白的活性、高级结构及功能都有重要的影响。
酵母表达的糖蛋白不同于哺乳动物表达的杂合型或复杂型糖蛋白，而是高甘露糖型或过度甘露糖化糖蛋白。
在前期成功敲除毕赤酵母α-1,6-甘露糖转移酶(Och1p)基因、阻断毕赤酵母过度糖基化，获得毕赤酵母过度糖基化缺陷菌株GJK01 (ura3、och1) 的基础上，通过表达不同物种来源的α-1,2-甘露糖苷酶I (MDSI) 的活性区与酵母自身定位信号的融合蛋白，并通过DSA-FACE (基于DNA测序仪的荧光辅助糖电泳) 分析筛选报告蛋白 HSA/GM-CSF (人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白) 的糖基结构，发现当编码酿酒酵母α-1,2-甘露糖苷酶(MnsI) 基因的内质网定位信号与带有完整C-端催化区的拟南芥MDSI基因融合表达时，毕赤酵母工程菌株能够合成Man5GlcNAc2哺乳动物甘露糖型糖蛋白。
这为在酵母体内合成类似于哺乳动物杂合型或复杂型糖基化修饰的糖蛋白奠定了基础。
【期刊名称】生物工程学报【年(卷),期】2011(027)001【总页数】10【关键词】关键词: α-1,2-甘露糖苷酶I，Man5GlcNAc2，糖基化，毕赤酵母，甘露糖型近年来，药用蛋白市场的需求越来越大，在这些蛋白中糖蛋白占绝大多数，例如各种治疗性抗体、细胞因子等。
糖蛋白是糖基与蛋白共价相连构成的结合蛋白，糖蛋白的糖基与蛋白的功能、稳定性均有着密切的联系[1]。
糖蛋白根据糖基和蛋白质的连接方式不同，可分为2大类，即O-连接和N-连接糖蛋白。
其中，对N-连接糖蛋白的糖基化修饰研究得比较透彻，其修饰序列极端保守，即在Asn-X-Thr/Ser (X为除Pro外的任意氨基酸) 的Asn残基上。
目前重组糖蛋白药物的生产主要是哺乳动物细胞表达系统。
但该系统培养成本高、生产周期长[2]。
因此，利用酵母细胞来替代哺乳动物细胞生产重组蛋白药物已日益受到人们的重视。
酵母表达系统具有原核细胞系统生长速度快、便于基因操作和可工业化大规模培养等优点，同时又具有真核细胞大部分的翻译后加工修饰能力，因而广泛用于各种蛋白的表达[3]。
巴斯德毕赤酵母属于甲基营养型酵母，是第2代酵母表达系统的代表，作为成熟的表达系统，它还具有表达量高、较稳定、可分泌、易纯化及成本低等优点[4-7]，但目前主要应用于非糖蛋白的生产，这是因为当毕赤酵母表达系统用于多数重组糖蛋白药物的生产时，还存在一些问题，其中最关键的是毕赤酵母对糖蛋白的过度糖基化修饰，即形成不同于哺乳动物细胞的杂合型或复杂型糖基结构，而是高甘露糖型糖基或过度糖基化结构[8-9]，这种高甘露糖型糖基结构的糖蛋白在人体中容易被清除、半衰期较短、且易产生较高的免疫原性；同时对某些蛋白过度的糖基化修饰也会造成产物的分子量不均一，或者一些活性位点的遮蔽[10]，影响其功能和活性。
这些都限制了毕赤酵母在糖蛋白类药物生产方面的应用。
酵母和哺乳动物N-糖基化修饰途径在内质网中的起始步骤都是相同的，在内质网中，在寡糖转移酶作用下，Glc3Man9GlcNAc2被连接到新生肽链的专一序列上，即Asn-X-Thr/Ser的Asn残基上，随后在葡萄糖苷酶Ⅰ、Ⅱ和内质网甘露糖苷酶 I (MnsI)的作用下，蛋白的糖基最终被加工成 Man8GlcNAc2糖基，并转运至高尔基体。
在高尔基体内，酵母和哺乳动物糖基修饰过程明显不同，在酵母高尔基体中，由于存在一个关键起始酶α-1,6-甘露糖转移酶(Och1p)，Man8GlcNAc2糖基在它的作用下接受第一个α-1,6-甘露糖，形成Man9GlcNAc2糖基，该糖基是α-1,2、α-1,3甘露糖转移酶及一些磷酸甘露糖转移酶的底物，在这些甘露糖转移酶的作用下，蛋白的糖基可以被加至数十至上百个甘露糖，形成高甘露糖，发生过度糖基化修饰[10]；而在哺乳动物高尔基体内，蛋白的糖基在α-1,2甘露糖苷酶I (MDSI)的作用下，Man8GlcNAc2糖基会被剪切去除3个α-1,2连接的甘露糖，而形成Man5GlcNAc2糖基，这一糖基是哺乳动物细胞合成杂合型和复杂型糖基的前体，也是哺乳动物表达的甘露糖型糖蛋白的主要糖型，如牛核糖核酸酶B (Ribonuclease B)。
而这种糖基在酵母中并不存在，所以这一结构也是酵母和哺乳动物N-糖基化修饰过程的“分界线”。
我们前期已经敲除了Och1这一形成高甘露糖型糖基结构的关键酶基因，获得了过度糖基化缺失的毕赤酵母菌株GJK01 (ura3、och1)[11]。
本研究拟通过选择不同物种来源MDSI及其定位信号，并实现其在GJK01 (ura3、och1) 中的融合表达，以获得从酵母糖基向哺乳动物糖基改造的第一个关键糖型，即Man5GlcNAc2结构。
1 材料与方法1.1 材料1.1.1 质粒和菌株α-1,6-甘露糖转移酶 (Och1p) 基因缺陷的毕赤酵母 GJK01 (ura3、och1) 菌株由本室构建。
HSA/GM-CSF表达载体pHIL-D2/HSA/GM-CSF、PGE1203-URA3 均由本室构建保存[11]。
载体pPICZαA、pIB2、毕赤酵母GS115均购自Invitrogen公司。
HepG2细胞由本室保存。
1.1.2 试剂和仪器培养基和试剂：酵母抽提物、蛋白胨均为Oxoid公司产品；无氨基酸酵母氮源(Yeast nitrogen base without amino acids，YNB) 为Difco公司产品；所用限制性内切酶、T4 DNA连接酶和去磷酸化酶(CIAP) 均为大连宝生物工程有限公司产品；牛核糖核酸酶B (RNase B)、氢化硼氰化钠(NaBH3CN) 均为Sigma产品；Sephadex-G10 柱色谱填料(AB)；DNA 3100 测序仪(ABI)；8-氨基芘基-1,3,6-三磺酸(8-aminopyrene-1,3,6-trisulfonate，APTS) (Molecular probes)；碳黑柱 (Alltech associates)。
1.2 α-1,2-甘露糖苷酶 (MDSI) 表达载体的构建1.2.1 PGE-URA3-GAP1载体构建GAP启动子基因的获取：以pGAP01和pGAP02为引物，以毕赤酵母表达载体pIB2为模板，PCR扩增500 bp GAP启动子基因片段；CY CTT终止子基因的获取：以pCYCTT01和pCYCTT02为引物，以毕赤酵母表达载体pPICZαA为模板，PCR扩增300 bp的CYCTT终止子基因片段；然后通过引物pGAP01和pCYCTT02，利用PCR方法融合GAP和CYCTT，获得GAP表达盒，片段大小约800 bp。
利用Kpn Ⅰ/XbaⅠ酶切该片段，并与经过同样酶切的pGE1203-URA3进行连接，获得含GAP表达盒的载体pGEURA3-GAP。
利用一对互补配对的寡核苷酸引物1203MSC5、1203MSC3，退火形成双链，构建至pGEURA3-GAP载体中，获得含有多克隆位点Ssp I-Sse838 I-Swa I 的载体，命名为 pGEURA3-GAP1载体。
1.2.2 载体PGE-URA3-GAP1-signal-MDSI的构建定位信号的克隆：以酿酒酵母基因组为模板，以pMnsI5、pMnsI3为引物，PCR扩增90 bp的MnsI定位信号，以pSCsec12-5、pSCsec12-3为引物，PCR扩增300 bp 的Sec12定位信号；以乳酸克鲁维酵母基因组为模板，以pKLsec12-5、pKLsec12-3为引物，PCR扩增300 bp的Sec12定位信号。
将扩增得到的3种定位信号基因片段分别用Sse8387Ⅰ/SspⅠ酶切，构建至同样酶切的pGE-URA3-GAP1载体中，获得3种pGE-URA3-GAP1-signal载体。
MDSI基因的克隆：从人的肝癌细胞HepG2中提取总RNA，以pHmdsi(Δ185)、pHMDSI3为引物，RT-PCR扩增1 800 bp人源的编码缺失N端185个氨基酸但包含完整C端催化区的MDSI基因片段，记为HMDSI(Δ185)；从植物拟南芥叶片中提取总RNA，以pATMDSI5(Δ48)、pATMDSI3为引物，RT-PCR扩增1 700 bp拟南芥的编码缺失N端48个氨基酸但包括完整C端催化区的MDSI基因片段，记为ATMDSI(Δ48)。
利用Sse8387Ⅰ/SwaⅠ酶切位点将2种基因片段分别构建至3种pGE-URA3-GAP1-signal载体中，获得 6种载体 PGE-URA3-GAP1-signal-MDSI。
1.2.3 表达载体PGE-URA3-PNOI-GAP-signal-MDSI的构建为了实现MDSI表达载体在酵母基因组中的定点整合，本研究选取了磷酸甘露糖转移酶PNO1基因开放读码框ORF上游同源序列作为整合位点。
因此，以毕赤酵母GS115基因组为模板，以PNOI5-5、Pno为引物，PCR扩增PNO1基因ORF上游序列，片段大小为1 000 bp左右，电泳回收，然后，再以回收产物与PNOI3-3为引物，以毕赤酵母GS115基因组为模板，PCR扩增2 000 bp左右基因片段，电泳回收，回收产物利用KpnⅠ/XbaⅠ酶切位点克隆至pGE-URA3-GAP载体中，获得pGE-URA3-PNOI载体；NotⅠ酶切6个基因载体PGE-URA3-GAP-signal-MDSI，将获得的带有MDSI基因的表达盒克隆到限制性内切酶NotⅠ酶切并 CIAP去磷酸化的pGE-URA3-PNOI载体中，获得表达载体 PGEURA3-PNOI-GAP1-signal-MDSI。
1.3 HSA/GM-CSF (His6-tag) 在过度糖基化缺陷菌GJK01 (ura3、och1) 中的表达及鉴定制备GJK01 (ura3、och1) 的电转化感受态细胞，将pHIL-D2/HSA/GM-CSF(His6-tag) 以限制性内切酶NotⅠ线性化后电转入感受态细胞中，将电击后的菌液涂布于含有尿嘧啶和精氨酸的MD 培养基(YNB 1.34%，生物素4×10−5%，葡萄糖2%，琼脂1.5%，精氨酸100 μg/mL，尿嘧啶100 μg/mL) 上。
5~8 d后，随机挑取克隆接种到2 mL YPD培养基中，25 ℃培养48 h后，以5%的接种量接种到BMGY培养基中，24 h后加入0.5%甲醇进行诱导表达，每12 h补加1次甲醇，诱导72 h后离心取上清，上清用于融合蛋白的表达分析，筛选HSA/GM-CSF(His6-tag)在缺陷菌GJK01 (ura3、och1) 中的表达株。