pPIC6 B酵母表达载体

表1.2 Invitrogen公司开发的常见表达载体
Table 1.2 Pichia pastoris expression vectors provided by Invitrogen.
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，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。

目前所采用的表达载体均为穿梭质粒，先在大肠杆菌保存、复制、扩增，然后线性化后被导入宿主酵母细胞。

常用的表达载体包括胞内表达载体及分泌表达载体（表1.2），主要是由启动子、终止子、外源基因克隆位点、选择标记、报告基因和复制起点等元件构成，分泌型载体还需要信号肽序列，如pPICZα、pPIC6α 和pPIC9K 的α-factor序列。

胞内表达适宜于不含二硫键的非糖基化蛋白，其表达水平较胞外高，但产物纯化较为复杂。

而需要翻译后修饰的蛋白，如抗体、细胞因子等一般选择胞外分泌表达。

蛋白被直接分泌到培养基中，而且酵母自身分泌的蛋白很少，更利于下游工作，也有助于形成糖基化的有活性天然构象[32, 90]。

His4组氨酸脱氢酶位点。

毕赤酵母表达载体及宿主菌介绍由于甲醇营养型酵母菌体内无天然质粒，所以携带外源基因的重组体必须整合于染色体中才能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。

如果是分泌型表达载体，在多克隆位点的前面，外源基因的5’端和启动子的3’端之间插人了分泌作用的信号肽序列。

在这个分泌信号的引导下，外源蛋白在内质网和高尔基体中经修饰和加工后能够由胞内转移至胞外，将成熟的蛋白质分泌到细胞外。

为方便于操作，通常表达载体都是穿梭质粒。

表达载体与酵母染色体有单交换和双交换整合2种方式，单交换整合时，或插入A0X1位点，或插入his位点。

有文献报道，以his4作为整合位点时，染色体突变株与表达盒间存在基因转换，偶而可使Laz表达盒丢失，故AOX1位点更好。

一般认为，单交换转化效率比双交换效率高，且易得到多拷贝整合，其发生机制可能是重复单交换引起的。

携带外源基因的表达载体可通过电穿孔、原生质体生成法或全细胞法转化酵母细胞。

甲醇酵母转化和大肠埃希氏菌转化不同之处是前者较为复杂。

对于大肠埃希氏菌而言，只要把重组表达载体导入细胞体内即可。

因其载体上携带有自身复制原点，可随染色体复制而复制，重组表达载体能够稳定存在。

在甲醇酵母系统中，所有的表达载体均不含酵母复制原点。

这就是说，导入酵母体内的重组表达载体只有和酵母染色体上的同源区发生重组，整合到染色体上，外源基因才能够稳定存在，外源蛋白才能得到稳定表达，这种整合的转化子一旦形成就非常稳定。

如果转化后的重组表达载体未能整合到染色体上，而是以游离的附加体形式存在，这种转化子就不稳定，重组表达载体极易丢失。

毕赤酵母表达的培养基配制［5］2.1 LB（Luria－Bertani）培养基：Trypton l％Y east Extract 0.5％NaCl l％PH 7.0制作平板时加入2％琼脂粉。

121℃高压灭菌20min。

可于室温保存。

用于培养pPICZαA原核宿主菌TOP10F’时可加入Zeocin 25ug / ml。

2.2 LLB（Low Salt LB）培养基：Trypton l％Y east Extract 0.5％NaCl 0.5％PH 7.0制作平板时加入2％琼脂粉。

121℃高压灭菌20min。

可于室温保存数月。

用于培养pPICZαA 原核宿主菌TOP10F’时，加入Zeocin 25ug / ml，可以4℃条件下保存1～2周。

2.3 YPD （又称YEPD）Y east Extract Peptone Dextrose Medium，（Y east Extract Peptone Dextrose Medium，酵母浸出粉/胰蛋白胨/右旋葡萄糖培养基）Trypton 2%dextrose (glucose) 2%+agar 2%+Zeocin 100 μg/ml液体YPD培养基可常温保存；琼脂YPD平板在4℃可保存几个月。

加入Zeocin 100ug / ml，成为YPDZ培养基，可以4℃条件下保存1～2周。

2.4 YPDS + Zeocin 培养基（Y east Extract Peptone Dextrose Medium）：yeast extract 1%peptone 2%dextrose (glucose) 2%sorbitol （山梨醇）1 M+agar 2%+ Zeocin 100 μg/ml不管是液体YPDS培养基，还是YPDS + Zeocin 培养基，都必须存放4℃条件下，有效期1～2周。

2.5 MGYMinimal Glycerol Medium （最小甘油培养基）（34%YNB；1%甘油；4*10-5%生物素）。

pPICZ A, B, and CPichia expression vectors for selection onZeocin™ and purification of recombinant proteins Catalog no. V190-20Rev. Date: 7 July 2010Manual part no. 25-0148MAN00000034User ManualiiTable of ContentsKit Contents and Storage (iv)Accessory Products (v)Introduction (1)Overview (1)Methods (3)Cloning into pPICZ A, B, and C (3)Pichia Transformation (9)Expression in Pichia (13)Purification (15)Appendix (17)Recipes (17)Zeocin™ (19)Map and Features of pPICZ A, B, and C (21)Lithium Chloride Transformation Method (23)Construction of In Vitro Multimers (24)Technical Support (32)Purchaser Notification (33)References (34)iiiKit Contents and StorageContents The following components are included with Catalog no. V190–20. Note that thepPICZ expression vectors are supplied in suspension.Component QuantityCompositionpPICZ A Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ B Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ C Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0GS115/pPICZ/lacZ Positive1 stab --Control strainShipping/Storage The components included with Catalog no. V190–20 are shipped on wet ice.Upon receipt, store as directed below.For long-term storage of your positive control stab strain, we recommendpreparing a glycerol stock immediately upon receipt and storing at –80°C.Component ShippingStorage pPICZ A Expression Vector Wet ice Store at –20°CpPICZ B Expression Vector Wet ice Store at –20°CpPICZ C Expression Vector Wet ice Store at –20°CGS115/pPICZ/lacZ positive control strain Wet ice Store at 4°CivAccessory ProductsAdditional ProductsThe products listed in this section are intended for use with the pPICZ vectors.For more information, visit our web site at or contactTechnical Support (page 32).Product QuantityCatalogno. X-33 Pichia strain 1 stab C180-00GS115 Pichia strain 1 stab C181-00KM71H Pichia strain 1 stab C182-00SMD1168H Pichia strain 1 stab C184-00pPICZα A, B, and C 20 μg each V195-20pPIC6α A,B, and C 20 μg each V215-20pPIC6 A, B, and C 20 μg each V210-20pPIC6 Starter Kit 1 kit K210-01Original Pichia Expression Kit 1 kit K1710-01EasySelect™Pichia Expression Kit 1 kit K1740-01Pichia EasyComp™ Transformation Kit 1 kit K1730-01Pichia Protocols 1 book G100-01PureLink™ Gel Extraction Kit 50 preps250 prepsK2100–12K2100–25S.N.A.P ™ Gel Purification Kit 25 preps K1999–25PureLink™ Quick Plasmid Miniprep Kit 50 preps250 prepsK2100–10K2100–11PureLink™ HiPure Plasmid Midiprep Kit 25 preps50 prepsK2100–04K2100–13One Shot® TOP10 (chemically competent E. coli) 10 reactions20 reactionsC4040–10C4040–03One Shot® TOP10 Electrocompetent E. Coli 10 reactions20 reactionsC4040-50C4040-52TOP10 Electrocomp™ Kits 20 reactions C664–55Positope™ Control Protein 5 μg R900-50CIAP (Calf Intestinal Alkaline Phosphatase) 1,000 units 18009–019T4 DNA Ligase 100 units500 units15224–01715224–025Zeocin™ 1g5 gR250-01R250-05β-Gal Assay Kit 1 kit K1455-01β-Gal Staining Kit 1 kit K1465-01E-Gel® Agarose Gels E-Gel® Agarose Gels are bufferless, pre-cast agarose gels designed for fast, convenient electrophoresis of DNA samples. E-Gel® agarose gels are available in different agarose percentage and well format for your convenience.For more details on these products, visit our web site at or contact Technical Support (page 32).Continued on next pagevAccessory Products, ContinuedZeocin™Zeocin™ may be obtained from Invitrogen (see above). For your convenience, the drug is prepared in autoclaved, deionized water and available in 1.25 ml aliquotsat a concentration of 100 mg/ml. The stability of Zeocin™ is guaranteed for sixmonths if stored at –20°C.Detection of Fusion Protein A number of antibodies are available from Invitrogen to detect expression ofyour fusion protein from the pPICZ vector. Horseradish peroxidase (HRP)-conjugated antibodies allow one-step detection in Western blots usingcolorimetric or chemiluminescent detection methods. The amount of antibodysupplied is sufficient for 25 Western Blots.Antibody Epitope Catalogno.Anti-myc R950–25 Anti-myc-HRPDetects the 10 amino acid epitopederived from c-myc (Evans et al., 1985):EQKLISEEDLR951–25Anti-His(C-term) R930–25Anti-His(C-term)-HRPDetects the C-terminal polyhistidine(6xHis) tag (requires the free carboxylgroup for detection) (Lindner et al., 1997):HHHHHH-COOHR931–25Purification of Fusion Protein The polyhistidine (6xHis) tag allows purification of the recombinant fusionprotein using metal-chelating resins such as ProBond™. Ordering information forProBond™ resin is provided below.Product QuantityCatalogno. ProBond™ Purification System 6 purifications K850–01ProBond ™ Purification System with Anti-myc-HRP Antibody1 Kit K852–01ProBond ™ Purification System with Anti-His(C-term)-HRP Antibody1 Kit K853–01ProBond™ Nickel-Chelating Resin 50 ml150 mlR801–01R801–15Purification Columns 50 each R640–50viIntroductionOverviewIntroduction pPICZ A, B, and C are 3.3 kb expression vectors used to express recombinantproteins in Pichia pastoris. Recombinant proteins are expressed as fusions to aC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.The vector allows high-level, methanol inducible expression of the gene ofinterest in Pichia, and can be used in any Pichia strain including X33, GS115,SMD1168H, and KM71H. pPICZ contains the following elements:•5′ fragment containing the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989;Tschopp et al., 1987a)•Zeocin™ resistance gene for selection in both E. coli and Pichia (Baron et al.,1992; Drocourt et al., 1990)•C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis)tag for detection and purification of a recombinant fusion protein (if desired)•Three reading frames to facilitate in-frame cloning with the C-terminalpeptideReference Sources The pPICZ A, B, and C expression vectors may be used with the Original Pichia Expression Kit, and are included in the EasySelect™Pichia Expression Kit (see page v for ordering information). Additional general information about recombinant protein expression in Pichia pastoris is provided in the manuals for the Original Pichia Expression Kit and the EasySelect™Pichia Expression Kit. For more information about the Original Pichia Expression Kit, the EasySelect™Pichia Expression Kit, or their manuals, visit our web site at or contact Technical Support (page 32).More detailed information and protocols dealing with Pichia pastoris may also be found in the following general reference:Higgins, D. R., and Cregg, J. M. (1998) Pichia Protocols. In Methods in Molecular Biology, Vol. 103. (J. M. Walker, ed. Humana Press, Totowa, NJ) (see page v for ordering information).Recommended Pichia Host Strain We recommend using the X-33 Pichia strain as the host for expression of recombinant proteins from pPICZ. Other Pichia strains including GS115, KM71H, and SMD1168H are suitable. The X-33 Pichia strain and other strains are available from Invitrogen (see page v for ordering information). The X-33 Pichia strain has the following genotype and phenotype:Genotype: Wild-typePhenotype: Mut+1Overview, ContinuedExperimental Overview The following table describes the basic steps needed to clone and express your gene of interest in pPICZ.Step Action1 Propagate pPICZ A, B, and C by transformation into a rec A, end A1E. coli strain such as TOP10, DH5 , or JM109.2 Develop a cloning strategy and ligate your gene into one of the pPICZvectors in frame with the C-terminal tag.3 TransformintoE. coli and select transformants on Low Salt LB platescontaining 25 μg/ml Zeocin™.4 Analyze 10–20 transformants by restriction mapping or sequencing toconfirm in-frame fusion of your gene with the C-terminal tag.5 Purify and linearize the recombinant plasmid for transformation intoPichia pastoris.6 TransformyourPichia strain and plate onto YPDS plates containing the appropriate concentration of Zeocin™.7 Select for Zeocin™-resistant transformants.8 Optimize expression of your gene.9 Purify your fusion protein on metal-chelating resin (i.e. ProBond™).Continued on next page2MethodsCloning into pPICZ A, B, and CIntroduction The pPICZ vector is supplied with the multiple cloning site in three readingframes (A, B, and C) to facilitate cloning your gene of interest in frame with theC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.Use the diagrams provided on pages 5–7 to help you design a strategy to cloneyour gene of interest in frame with the C-terminal peptide. Generalconsiderations for cloning and transformation are discussed in this section.General Molecular Biology Techniques For assistance with E. coli transformations, restriction enzyme analysis, DNA biochemistry, and plasmid preparation, refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).E. coli Strain Many E. coli strains are suitable for the propagation of the pPICZ vectorsincluding TOP10, JM109, and DH5 . We recommend that you propagate thepPICZ vectors in E. coli strains that are recombination deficient (rec A) andendonuclease A deficient (end A).For your convenience, TOP10 E. coli are available as chemically competent orelectrocompetent cells from Invitrogen (page v).Transformation Method You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.Maintenance of Plasmids The pPICZ vectors contain the Zeocin™ resistance (Sh ble) gene to allow selection of the plasmid using Zeocin™. To propagate and maintain the pPICZ plasmids, we recommend using the following procedure:e 10 ng of your vector to transform a rec A, end A E. coli strain like TOP10,DH5 , JM109, or equivalent (see above).2.Select transformants on Low Salt LB plates containing 25 μg/ml Zeocin™ (seepage 17 for a recipe).3.Prepare a glycerol stock from each transformant containing plasmid forlong-term storage (see page 8).Continued on next page3Cloning into pPICZ A, B, and C, ContinuedGeneral Considerations The following are some general points to consider when using pPICZ to express your gene of interest in Pichia:•The codon usage in Pichia is believed to be similar to Saccharomyces cerevisiae.•Many Saccharomyces genes have proven to be functional in Pichia.•The premature termination of transcripts because of "AT rich regions" has been observed in Pichia and other eukaryotic systems (Henikoff & Cohen, 1984; Irniger et al., 1991; Scorer et al., 1993; Zaret & Sherman, 1984). If you have problems expressing your gene, check for premature termination by northern analysis and check your sequence for AT rich regions. It may be necessary to change the sequence in order to express your gene (Scorer et al., 1993).•The native 5´ end of the AOX1 mRNA is noted in the diagram for each multiple cloning site. This information is needed to calculate the size of the expressed mRNA of the gene of interest if you need to analyze mRNA for any reason.Cloning Considerations For proper initiation of translation, your insert should contain an initiation ATG codon as part of a yeast consensus sequence (Romanos et al., 1992). An example of a yeast consensus sequence is provided below. The ATG initiation codon is shown underlined.(G/A)NNATG GTo express your gene as a recombinant fusion protein, you must clone your gene in frame with the C-terminal peptide containing the c-myc epitope and the polyhistidine tag. The vector is supplied in three reading frames to facilitate cloning. Refer to the diagrams on pages 5–7 to develop a cloning strategy.If you wish to express your protein without the C-terminal peptide, be sure to include a stop codon.Construction of Multimeric Plasmids pPICZ A, B, and C contain unique Bgl II and Bam H I sites to allow construction of plasmids containing multiple copies of your gene. For information on how to construct multimers, refer to pages 24–31.Continued on next page4Multiple CloningSite of pPICZ A Below is the multiple cloning site for pPICZ A. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ A from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGTTCCTCAG TTCAAGTTGG GCACTTACGA GAAGACCGGT CTTGCTAGAT TCTAATCAAG AGGATGTCAG AATGCCATTT GCCTGAGAGA TGCAGGCTTC ATTTTTGATA CTTTTTTATTTGTAACCTAT ATAGTATAGG ATTTTTTTTG TCATTTTGTT 1218Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTTAGCCT TAGACATGAC AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT GGGCCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´polyadenylation sitePolyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Apa I 104210981158871931991Continued on next pageMultiple CloningSite of pPICZ B Below is the multiple cloning site for pPICZ B. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ B from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1216Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATTATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT TCTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Xba I 104010961156871931991Continued on next pageMultiple CloningSite of pPICZ C Below is the multiple cloning site for pPICZ C. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ C from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1217Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming siteTGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT ACGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho I Sac II Not I Sna B I 104110971157871931991Continued on next pageE. coli Transformation Transform your ligation mixtures into a competent rec A, end A E. coli strain(e.g. TOP10, DH5, JM109) and select on Low Salt LB agar plates containing25 μg/ml Zeocin™ (see below). Note that there is no blue/white screening for the presence of insert with pPICZ A, B, or C. Once you have obtained Zeocin™-resistant colonies, pick 10 transformants and screen for the presence and orientation of your insert.Important To facilitate selection of Zeocin™-resistant E. coli, the salt concentration of the medium must remain low (<90 mM) and the pH must be 7.5. Prepare Low Salt LB broth and plates using the recipe in the Appendix, page 17.Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug.C We recommend that you sequence your construct to confirm that your gene is in the correct orientation for expression and cloned in frame with the C-terminal peptide (if desired). Refer to the diagrams on pages 5–7 for the sequences and location of the priming sites.Preparing a Glycerol Stock Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at –20°C.1.Streak the original colony out on an Low Salt LB plate containing 25 μg/mlZeocin™. Incubate the plate at 37°C overnight.2.Isolate a single colony and inoculate into 1–2 ml of Low Salt LB containing25 μg/ml Zeocin™.3.Grow the culture to mid-log phase (OD600 = 0.5–0.7).4.Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to acryovial.5.Store at –80°C.Plasmid Preparation Once you have cloned and sequenced your insert, generate enough plasmid DNA to transform Pichia (5–10 μg of each plasmid per transformation). We recommend isolating plasmid DNA using the PureLink™ Quick Plasmid Miniprep Kit or the PureLink™ HiPure Plasmid Midiprep Kit (page v), or CsCl gradient centrifugation.Once you have purified plasmid DNA, proceed to Pichia Transformation, next page.Pichia TransformationIntroduction You should now have your gene cloned into one of the pPICZ vectors. Yourconstruct should be correctly fused to the C-terminal peptide (if desired). Thissection provides general guidelines to prepare plasmid DNA, transform yourPichia strain, and select for Zeocin™-resistant clones.Zeocin™ Selection We generally use 100 μg/ml Zeocin™ to select for transformants when using the X-33 Pichia strain. If you are transforming your pPICZ construct into anotherPichia strain, note that selection conditions may vary. We recommendperforming a dose response curve to determine the appropriate concentration ofZeocin™ to use for selection of transformants in your strain.Method of Transformation We do not recommend spheroplasting for transformation of Pichia with plasmids containing the Zeocin™ resistance marker. Spheroplasting involves removal of the cell wall to allow DNA to enter the cell. Cells must first regenerate the cell wall before they are able to express the Zeocin™ resistance gene. For this reason, plating spheroplasts directly onto selective medium containing Zeocin™ does not yield any transformants.We recommend electroporation for transformation of Pichia with pPICZ A, B, or C. Electroporation yields 103 to 104 transformants per μg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, use the LiCl protocol on page 23 or the Pichia EasyComp™Transformation Kit available from Invitrogen (see below).PichiaEasyComp™Transformation Kit If you wish to perform chemical transformation of your Pichia strain with pPICZ A, B, or C, the Pichia EasyComp™ Transformation Kit is available from Invitrogen (see page v for ordering information). The Pichia EasyComp™ Transformation Kit provides reagents to prepare 6 preparations of competent cells. Each preparation will yield enough competent cells for 20 transformations. Competent cells may be used immediately or frozen and stored for future use. For more information, visit our web site at or contact Technical Support (page 32).Important Since pPICZ does not contain the HIS4 gene, integration can only occur at the AOX1 locus. Vector linearized within the 5´ AOX1 region will integrate by gene insertion into the host 5´ AOX1 region. Therefore, the Pichia host that you use will determine whether the recombinant strain is able to metabolize methanol (Mut+) or not (Mut S). To generate a Mut+ recombinant strain, you must use a Pichia host that contains the native AOX1 gene (e.g. X-33, GS115, SMD1168H). If you wish to generate a Mut S recombinant strain, then use a Pichia host that has a disrupted AOX1 gene (i.e. KM71H).Continued on next pageHis4 Host Strains Host strains containing the his4 allele (e.g. GS115) and transformed with thepPICZ vectors require histidine when grown in minimal media. Add histidine toa final concentration of 0.004% to ensure growth of your transformants.The pPICZ vectors do not contain a yeast origin of replication. Transformantscan only be isolated if recombination occurs between the plasmid and the Pichiagenome.Materials Needed You will need the following items:Note: Inclusion of sorbitol in YPD plates stabilizes electroporated cells as they appear tobe somewhat osmotically sensitive.•5–10 μg pure pPICZ containing your insert•YPD Medium•50 ml conical polypropylene tubes• 1 liter cold (4°C) sterile water (place on ice the day of the experiment)•25 ml cold (4°C) sterile 1 M sorbitol (place on ice the day of the experiment)•30°C incubator•Electroporation device and 0.2 cm cuvettes•YPDS plates containing the appropriate concentration of Zeocin™ (seepage 18 for recipe)Linearizing YourpPICZ ConstructTo promote integration, we recommend that you linearize your pPICZ constructwithin the 5′ AOX1 region. The table below lists unique sites that may be used tolinearize pPICZ prior to transformation. Other restriction sites are possible.Note that for the enzymes listed below, the cleavage site is the same for versionsA, B, and C of pPICZ. Be sure that your insert does not contain the restriction siteyou wish to use to linearize your vector.Enzyme Restriction Site (bp) SupplierSac I 209 ManyPme I 414 New England BiolabsBst X I 707 ManyRestriction Digest 1.Digest ~5–10 μg of plasmid DNA with one of the enzymes listed above.2.Check a small aliquot of your digest by agarose gel electrophoresis forcomplete linearization.3.If the vector is completely linearized, heat inactivate or add EDTA to stopthe reaction, phenol/chloroform extract once, and ethanol precipitate using1/10 volume 3 M sodium acetate and 2.5 volumes of 100% ethanol.4.Centrifuge the solution to pellet the DNA, wash the pellet with 80% ethanol,air-dry, and resuspend in 10 μl sterile, deionized water. Use immediately orstore at –20°C.Continued on next pagePreparation of Pichia for Electroporation Follow the procedure below to prepare your Pichia pastoris strain for electroporation.1. Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical tube at30°C overnight.2. Inoculate 500 ml of fresh medium in a 2 liter flask with 0.1–0.5 ml of theovernight culture. Grow overnight again to an OD600 = 1.3–1.5.3. Centrifuge the cells at 1500 × g for 5 minutes at 4°C. Resuspend the pelletwith 500 ml of ice-cold (0–4°C), sterile water.4. Centrifuge the cells as in Step 3, then resuspend the pellet with 250 ml ofice-cold (0–4°C), sterile water.5. Centrifuge the cells as in Step 3, then resuspend the pellet in 20 ml of ice-cold (0–4°C) 1 M sorbitol.6. Centrifuge the cells as in Step 3, then resuspend the pellet in 1 ml of ice-cold(0–4°C) 1 M sorbitol for a final volume of approximately 1.5 ml. Keep the cells on ice and use that day. Do not store cells.Transformation by Electroporation 1.Mix 80 μl of the cells from Step 6 (above) with 5–10 μg of linearized pPICZDNA (in 5–10 μl sterile water) and transfer them to an ice-cold (0–4°C)0.2 cm electroporation cuvette.2.Incubate the cuvette with the cells on ice for 5 minutes.3.Pulse the cells according to the parameters for yeast (Saccharomycescerevisiae) as suggested by the manufacturer of the specific electroporation device being used.4.Immediately add 1 ml of ice-cold 1 M sorbitol to the cuvette. Transfer thecuvette contents to a sterile 15 ml tube.5.Let the tube incubate at 30°C without shaking for 1 to 2 hours.6.Spread 50-200 μl each on separate, labeled YPDS plates containing theappropriate concentration of Zeocin™.7.Incubate plates for 2–3 days at 30°C until colonies form.8.Pick 10–20 colonies and purify (streak for single colonies) on fresh YPD orYPDS plates containing the appropriate concentration of Zeocin™.Continued on next pageGenerally, several hundred Zeocin™-resistant colonies are generated using theprotocol on the previous page. If more colonies are needed, the protocol may bemodified as described below. Note that you will need ~20, 150 mm plates withYPDS agar containing the appropriate concentration of Zeocin™.1. Set up two transformations per construct and follow Steps 1 through 5 ofthe Transformation by Electroporation protocol, page 11.2. After 1 hour in 1 M sorbitol at 30°C (Step 5, previous page), add 1 ml YPDmedium to each tube.3. Shake (~200 rpm) the cultures at 30°C.4. After 1 hour, take one of the tubes and plate out all of the cells by spreading200 μl on 150 mm plates containing the appropriate concentration ofZeocin™.5. Optional: Continue incubating the other culture for three more hours (for atotal of four hours) and then plate out all of the cells by spreading 200 μl on150 mm plates containing the appropriate concentration of Zeocin™.6. Incubate plates for 2–4 days at 30°C until colonies form.Mut Phenotype If you used a Pichia strain containing a native AOX1 gene (e.g. X-33, GS115,SMD1168H) as the host for your pPICZ construct, your Zeocin™-resistanttransformants will be Mut+. If you used a strain containing a deletion in theAOX1 gene (e.g. KM71H), your transformants will be Mut S.If you wish to verify the Mut phenotype of your Zeocin™-resistant transformants,you may refer to the general guidelines provided in the EasySelect™PichiaExpression Kit manual or the Original Pichia Expression Kit manual or topublished reference sources (Higgins & Cregg, 1998).You are now ready to test your transformants for expression of your gene ofinterest. See Expression in Pichia, next page.。

毕赤酵母表达系统步骤（参考Invitrogen公司说明书）：一、pPICZαA、B、C质粒以及DH5α菌株的保存1取0.5μl pPICZα A、B、C质粒，热击转化DH5α，在低盐LB（含有25μg/ml Zeocin）的平板上37℃培养过夜。

2挑取转化子，甘油保存。

二、载体构建1将目的基因构建到pPICZα载体上，转化DH5α，用Zeocin筛选转化子。

2提质粒酶切鉴定或PCR鉴定3载体测序测序可用α-Factor引物或5’AOX1引物，3’AOX1引物三、线性化DNA1提取足够量的质粒DNA（一次转化至少需要5-10μg质粒）2 酶切线性化10μg构建好的载体，同时酶切空载体做对照，根据载体选择线性化酶切位点（样品分管酶切），pPICZα载体在5’AOX1区域有三个酶切位点可选择：SacI、PmeI、BstXI3 取1-2μl酶切产物跑电泳，确定是否酶切完全；4 过柱纯化线性化质粒（用50μl EB洗脱）；四、线性化DNA的去磷酸化处理线性化质粒43μlCIAP Buffer 5μlCIAP酶2μl四、总体积为50μl的样品37℃ 1h，过柱纯化，用30μl ddH2O洗脱；五、感受态细胞的制备实验前准备：无抗性YPD平板一个、无抗生素液体YPD培养基，100μg/ml Zeocin YPD 平板和液体、50ml离心管两个、500ml预冷的无菌水、20ml 1M 山梨醇（灭菌预冷的），0.2cm预冷的电击杯；1YPD平板划线培养菌，30℃培养2-3d；250ml三角瓶中，加入5ml YPD，挑取酵母单菌落，30℃培养过夜；3吸取0.5ml菌液，加入至含有200ml新鲜YPD的1L三角瓶中，30℃，225rpm/min培养至OD值1.3-1.5；41500g，4℃离心5min收集菌体；540ml冰预冷的无菌水重悬沉淀；61500g，4℃,5min；730ml无菌水重悬；81500g，4℃,5min；910ml 1M 山梨醇重悬；101500g，4℃,5min；11加入1ml山梨醇，重悬冰上放置，直接做转化，或加入灭菌甘油每管80ul分装，冻存于-80℃(长时间保存会影响转化效率)；六、电击转化15-10μg线性化DNA（20μl<）与80ul上述感受态细胞混合，转移至预冷的0.2cm电击杯中（点击条件：电压1.5kV；电容25µF；电阻200Ω，电击时间为4～10msec）；2冰上放置5min3电击（按生产厂商提供的适合酵母用的参数）4迅速加入1ml预冷的1M 山梨醇，转移至1.5ml EP管中530℃静置培养1-2h（如果要增加存活率，获得更多的转化克隆，可在30℃静置培养1h后，加入1mlYPD培养基，30℃200rpm培养1h后取部分涂布与不同浓度抗生素的平板）6取50、100、200ul分别涂布于含有Zeocin的YPD平板，30℃培养2-10 d至有菌落出现；7如果要筛选多拷贝转化子，将转化克隆混合在一起，涂布在Zeocin 浓度为500、1000、2000μg/ml的YPD平板，培养2-3d。

毕赤酵母常用培养基与载体一、毕赤酵母表达常用载体：典型的巴斯德毕赤酵母表达载体载体包含醇氧化酶－1（AOX1）基因的启动子和转录终止子（5'AOX1和3'AOX1），它们被多克隆位点（MCS）分开，外源基因可以在此插入。

此载体还包含组氨醇脱氢酶基因（HIS4）选择标记及3'AOX1区。

当整合型载体转化受体时，它的5'AOX1和3'AOX1能与染色体上的同源基因重组，从而使整个载体连同外源基因插入到受体染色体上，外源基因在5'AOX1启动子控制下表达。

毕赤酵母本身不分泌内源蛋白，而外源蛋白的分泌需要具有引导分泌的信号序列。

而由89个氨基酸组成的酿酒酵母的分泌信号—α交配因子(α-factor)引导序列已经成功地引导了几种外源蛋白的分泌。

分泌表达载体主要有：pPIC9，pPIC9K，pHIL-S1，pPICZα A，pYAM75P等。

胞内表达载体主要有：pHIL-D2，pA0815，pPIC3K，pPICZ，pHWO10，pGAPZ，pGAPZa(Invitrogen)，pPIC3.5K等。

工程菌株Y11430，MG1003，GS115 （AOX1），KM71，SMD1168。

毕赤酵母宿主菌常用的有GS115和KM71两种，都具有HIS4营养缺陷标记。

其中，GS115茵株具有AOX1基因，是Mut＋，即甲醇利用正常型；而KM71菌株的AOX1位点彼ARG4基因插入，表型为Muts，即甲醇利用缓慢型，两种菌株都适用于一般的酵母转化方法。

多拷贝表达菌株的获得方式：与自主复制的质粒型表达载体不同，整合型表达载体的拷贝数可以有很大的变化。

含多拷贝外源基因的表达菌株合成蛋白的量也较多。

体内整合可通过高遗传霉素抗性，筛选可能的多拷贝插入；而体外整合可通过连接产生外源基因的串联插入。

多拷贝表达菌株的获得方式有两种：一种是利用SDS-PAGE 电泳、免疫杂交或菌落点杂交方法在大量的转化子中进行自然筛选。

酵母表达体系毕赤酵母是甲醇营养型，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和过氧化氢。

为避免过氧化氢的毒性，甲醛代谢主要在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。

由于醇氧化酶与O2 的结合率较低，因而毕赤酵母代偿性地产生大量的酶。

而调控产生醇氧化物酶的启动子也正是驱动外源基因在毕赤酵母中表达的启动子。

毕赤酵母含有两种醇氧化物酶，AOX1 AOX2。

细胞中大多数的醇氧化酶是AOX1 基因产物。

甲醇可紧密调节、诱导 AOX1 基因的高水平表达，为Mut+菌株，可占可溶性蛋白的30%以上。

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，通过这种甲醇利用缓慢表型可分离Muts 菌株。

毕赤酵母表达外源蛋白：分泌型和胞内表达。

利用含有α因子序列的分泌型载体即可。

翻译后修饰：酿酒酵母与毕赤酵母大多数为N-连接糖基化高甘露糖型，毕赤酵母中蛋白转录后所增加的寡糖链长度（平均每个支链8-14 个甘露糖残基）比酿酒酵母中的（50-150 个甘露糖残基）短得多。

菌株：GS115 （ Mut+， Muts）和 KM71（Muts）分泌型载体:pPICZα A,B,and C(5’AOX1启动子，紧密型调节，甲醇诱导表达，α分泌信号介导的分泌表达，Zeocin抗性基因，C端含有6XHis标签)胞内表达型载体：pPICZ A,B,and C，一：分子克隆1.设计引物分泌型载体图谱：见酵母表达说明书（p13-pPICZ A，p14-pPICZ B,p15-pPICZ C）2.PCR扩增基因PCR反应体系（50μl）模板DNA 1μlForward Primer（10μM）1μlReverse Primer（10μM）1μldNTP Mixture(各2mM): 4μl5×PrimerSTAR buffer（Mg2+ plus）10μlPrimerSTAR DNA Polymerase 0.5μlddHO up to 50μl2PCR 反应流程预变性98℃ 2min变性98℃ 10sec退火56℃ 10sec 30个循环延伸72℃ 30sec完全延伸72℃ 10min保存4℃3.双酶切及其回收双酶切反应体系（40μl）DNA(空载体或目的基因) 30μlBamHⅠ 1.5μlXholⅠ 1.5μl10×Buffer K 4.0μl4.酶连接首先利用1%的琼脂糖电泳将双酶切后的PCR产物和载体进行分离,并通过胶回收试剂盒回收，按照目的基因和空载体的碱基摩尔比在1:3--1:9之间，一共吸取目的基因和空载体的总体积为5μl，在加入等量的5μl DNA快速连接试剂盒SolutionⅠ，16℃连接4-6h。

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。

α文章编号:10083464(2003)01003704巴斯德毕赤酵母新型分泌表达载体构建韦宇拓,甘凤琼,苏华波,赵颖怡,汪嵘,黄日波(广西大学生物技术实验中心,广西南宁530005)摘要:巴斯德毕赤酵母分泌表达载体pP I C I NU 是利用来源于克鲁维酵母(K luy vero m y ces m arx ianus )的菊粉酶基因信号肽DNA 序列(ISP )构建的。

表达实验结果表明,带有分泌表达载体pP I C I NU 的Β-1,3-1,4葡聚糖酶基因重组菌,具有与带有表达载体pP I C 9K (带有Α因子信号肽)的Β-1,3-1,4葡聚糖酶基因重组菌相同的分泌效率。

关键词:巴斯德毕赤酵母;克鲁维酵母菊粉酶基因信号肽;分泌表达;Α因子信号肽中图分类号:Q 782 文献标识码:ACon struction of a novel secreti ng expression vectors for P ich ia p astorisW E I Yu tuo ,GAN Feng qi ong ,SU H ua bo ,ZHAO Y ing yi ,W AN G Rong ,HUAN G R i bo(B i o techno logy R esearch Cen ter ,Guangx i U n iversity ,N ann ing 530005,Ch ina )Abstract :T he secreting exp ressi on vecto r ,pP I C I NU con tain ing inu linase signal pep tide (ISP )from K luy vero m y ces s m a rx ianu ,of P ich ia p astoris w as con structed .T he exp ressi on resu lts show ed that the secreti on efficiency of the beta1,31,4glucanase of recom b inan t P .p astoris con tain ing p P I C I NU w as as h igh as that of recom b inan t P .p astoris harbo ring pP I C I 9K carrying Αfacto r p rep ro p ep tide .Key wards :P ich ia p astori ;signal pep tides of K luy vero m y ces s m a rx ianu inu linase ;secreting ex 2p ressi on ;Α-facto r p rep ro pep tide Bong 等研究中发现在克鲁维酵母(K luy vero m y ces m a rx ianus )中,大部分菊粉酶能被分泌到胞外,将其先导肽用在酿酒酵母中能促进多种外源蛋白分泌到胞外,分泌效率高于常用的Α因子信号肽[1]。

毕式酵母表达常见问题及解析点击次数：433 作者：佚名发表于：2008-08-27 15:20转载请注明来自丁香园1、问：我用毕式酵母表达一个27KD的蛋白，小量表达并做SDS－PAGE有一条类似三聚体的条带。

用大量表达，表达上清用镍柱进行亲和层析，在FPLC上可以看到一个大峰，但跑电泳却没有发现条带，为什么呢？请赐教参考见解：你在大量表达后有没有跑电泳检测?表达上清用镍柱亲和层析后洗脱液有没有电泳检测?你首先要做的是:1,确定你的蛋白确实有表达;2,亲和层析是表达的蛋白确实被洗脱下来了.然后再考虑FPLC的事.在FPLC上可以看到一个大峰，但跑电泳却没有发现条带，可能的原因有：1，上样时没有目的蛋白，可能是1)没表达2)亲和层析时没洗脱或是穿透了；2，目的蛋白结合在FP LC柱子上没被洗脱；3，目的蛋白穿透FPLC柱子，你在FPLC上看到的大峰可能是盐分峰；4，电泳时没跑好,目的蛋白没被检测到.FPLC大峰也可能是目的蛋白峰，它的吸收值有多少？不要看与基线相比的高度,要看吸收值，如果吸收值不高的话就是蛋白的浓度太低,浓缩后再跑电泳。

2、问：我现在做的是裂殖酵母表达，表达载体pesp-2,酵母菌珠sp-q01.说明只提供用化学转化法，但我转化了好几次都没有结果，是否也可以用电转化，在多克隆位点把质粒线性化？参考见解：其实无论是那种类型的酵母表达，转化方法基本是一制的。

化学转化法对酵母转化讲就不是很高，这里你可以用电转的。

查一下以前别人的讨论，看看电转化方法，效率提高了，筛选的希望就大些了由于筛选表达酵母的时间比较长，两个方案同时做应该来的及的至于线性化，对于电转是要做的步骤，这是为了后面定位重组整合。

3、问：我做毕赤酵母分泌表达,想问做阴性对照的是应该用加甲醇前的上清还是用一直不加甲醇摇瓶同样时间的上清呢?参考见解：对于阴性对照我一般都用转了不含基因的原始质粒（比如９K等）的菌作为对照，诱导过程中同样加入甲醇，诱导的时间均一样，每次样品都会有一个对照。