PPT精品文档---巴斯德毕赤酵母
巴斯德毕赤酵母(Pichia pastoris)表达系统综述27页PPT
营养要求简单，生长快速，适合高密度大规模培养， 很少产生有毒物质，毒性比细菌小，用甲醇不易染菌， 可以减少污染。
高的调控功能，可用于外源基因的表达调控。甲 醇营养型酵母表达系统以巴斯德毕赤酵母(Pichia pastoris)表达系统最为常用。
（3）裂殖酵母(Schizogenesis pombe) 表达系统
只能以分裂和产孢子的方式繁殖的一类酵母， 因此定名为裂殖酵母。与前面几种酵母相比， 它具有更多的与高等真核生物相似的特性： 线 粒体结构、 启动子结构、 转录机制和对蛋白2端 酰基化功能均更接近于哺乳类细胞， 因而正逐 渐成为研究真核细胞分子生物学的模式生物， 它作为外源基因表达系统也开始受到人们的关注。 目前， 已经有多种蛋白利用此系统进行了表达， 如人蛋白凝血因子)G、 细胞色素 V6:8、 人白细 胞介素 YL’D等。此系统表达的外源蛋白更接近 于它们的天然形式 。
的真核生物， 其全序列的测定已于 2019年完成 。 酿酒酵母难于高密度培养，分泌效率低，几乎不分泌分
子量大于30 kD 的外源蛋白质，也不能使所表达的外源 蛋白质正确糖基化，而且表达蛋白质的C 端往往被截短。 因此，一般不用酿酒酵母做重组蛋白质表达的宿主菌。 酿酒酵母本身含有质粒，其表达载体可以有自主复制型 和整合型两种。值得注意的是，酿酒酵母表达的外源蛋 白质往往被高度糖基化，糖链上可以带有40 个以上的 甘露糖残基，糖蛋白的核心寡聚糖链仅含有末端1, 3 甘 露糖，产物的抗原性明显增强。所以，酿酒酵母常常用 来制备亚单位疫苗(如默克乙肝疫苗、口蹄疫疫苗等)。
1993年，Philip Petroleum公司将毕赤酵母表达系统的专利卖给 Research Corporation Technologies公司，并委托Invitrogea公司 进行有关产品销售。
巴斯德毕赤酵母
巴斯德毕赤酵母表达系统点击认领巴斯德毕赤酵母表达系统是近十年发展起来的真核表达体系，是目前最为成功的外源蛋白表达系统之一，与现有的其它表达系统相比，巴斯德毕赤酵母在表达产物的加工、外分秘、翻译后修饰以及糖基化修饰等方面有明显的优势，现已广泛用于外源蛋白的表达。
编辑摘要巴斯德毕赤酵母表达系统 - 简介十年代巴斯德毕赤酵母曾被用于生产单细胞蛋白（SCP），有很好的发酵基础，菌体密度可达100g/L干重。
其生长培养液的组分包括无机盐、微量元素、生物素、氮源和碳源，廉价而无毒。
它能在以甲醇为唯一碳源的培养基中快速生长，其中醇氧化酶AOX——甲醇代谢途径的关键酶可达细胞可溶性蛋白的30%。
而在葡萄糖、甘油或乙醇作为碳源的培养细胞中则检测不到AOX。
AOX的合成是在转录水平调控的。
其基因启动子具有明显的调控功能，可用于调控外源基因的表达。
此调控作用是由一般碳源抑制/解抑制及碳源特殊诱导又重机制控制的。
外源基因在甲醇以外的碳源中处于非表达状态，而在培养液中加入甲醇后，外源基因即被诱导表达。
巴斯德毕赤酵母中存在着一种称为微体的细胞器，其中大量合成过氧化物酶，因此也称为过氧化物酶体。
合成的蛋白质贮存于微体中，可免受蛋白酶的降解，且不对细胞产生毒害。
巴斯德毕赤酵母表达系统 - 毕赤酵母表达系统的特点自从1987年Cregg等首次用巴斯德毕赤酵母（Pichia pastoris）作为宿主表达外源蛋白以来,作为一种新的高效的表达系统，毕赤酵母越来越引起人们的重视，到1995年，已有四十多种外源蛋白在该宿主菌中获得表达。
而最近几年每年报道的在毕赤酵母中表达的外源基因就有几十种，且一年比一年多，与其它表达系统相比，毕赤酵母表达系统具有以下优势：1）含有特有的强有力的AOX（醇氧化酶基因）启动子，用甲醇可严格地调控外源基因的表达；2）表达水平高，即可在胞内表达，又可分泌型表达。
毕赤酵母中，报道的最高表达量为破伤风毒素C为12g/l，一般大于1g/l。
6毕赤酵母介绍
毕赤酵母及级胰岛素——优思灵巴斯德毕赤酵母(P／ch／a pastor／s)表达系统是目前最优秀、应用最广泛的外源基因表达系统之一，它不但克服了大肠杆菌表达系统不能表达结构复杂的蛋白质、表达的蛋白多形成不溶性包涵体、背景蛋白多、表达产量低等缺陷，弥补了哺乳类细胞、昆虫细胞表达系统操作复杂、表达水平低、产业化生产造价昂贵的不足，还具有其他酵母表达系统无法比拟的优越之处。
酵母作为一种最简单的单细胞真核生物, 兼有原核生物和真核生物的某些优点。
毕赤酵母生活史与酿酒酵母十分相似。
这使得毕赤酵母的发酵工程部分可与酿酒酵母相同, 从而节省了重新购臵设备和进行新技术探索的巨额花费, 这是用其他方法进行大规模商品化的外源蛋白生产所不能比拟的优势。
现将毕赤酵母表达系统优点概括如下:①毕赤酵母更接近高等真核细胞, 不含内毒素和其他有害物质, 使用安全;②同酿酒酵母一样, 繁殖速度快, 对培养条件要求低, 培养基价廉, 能进行高密度培养, 且能耐受较高的液体静压, 便于工业化生产;②该系统的载体能与核基因组进行整合, 因此构建的菌株较稳定, 一般不会在传代过程中出现丢失现象;③在毕赤酵母中表达的蛋白既可存在于胞内, 又可在分泌信号作用下将蛋白分泌至胞外;⑤具有强有力的易控制的乙醇氧化酶基因(AOX1)或磷酸甘油酸脱氢酶( GAP) 基因启动子作为外源基因启动子, 可对外源蛋白的表达进行严格调控;⑥作为真核表达系统, 可对表达的外源蛋白进行翻译后的加工和修饰, 即二硫键的形成、脂肪的酰化、蛋白磷酸化、折叠、信号序列加工、N糖基化、O糖基化, 从而使目的蛋白具有所应有的生物学活性;⑦外源蛋白表达量高, 同时自身分泌的背景蛋白少, 表达产物易于纯化;⑧和酿酒酵母相比, 毕赤酵母中加到翻译蛋白上的糖链长度平均每条侧链为8～14个甘露糖残基, 比酿酒酵母每条侧链平均50～150个甘露糖残基短得多。
毕赤酵母极少出现过渡糖基化, 可避免外源蛋白生产过强的免疫原性或因糖链过长干扰外源蛋白正确折叠而影响其功能, 而且糖链中不含具有致敏作用的α21, 32甘露糖, 使其使用更加安全。
毕赤酵母菌
——毕赤酵母
（Pichia.pastoris）
by：文慧霖、裴颖
· 毕赤酵母的介绍 · 毕赤酵母的形态特征 · 毕赤酵母的生产过程 · 毕赤酵母的优缺点 · 毕赤酵母的开发利用
毕赤酵母菌
——全称巴斯德毕赤酵母，是甲醇营养 型酵母中的一类能够利用甲醇作为唯一 碳源和能源的酵母菌。与其它酵母一样， 在无性生长期主要以单倍体形式存在， 当环境营养限制时，常诱导2个生理类 型不同的接合型单倍体 细胞交配，融 合成双倍体。
毕赤酵母结构图
毕赤酵母显微图
优缺点
Pichia.pastoris酵母菌体内无 天然质粒，所以表达载体需与宿 主染色体发生同源重组，将外源 基因表达框架整合于染色体中以 实现外源基因的表达。包括启动 子、外源基因克隆位点、终止序 列、筛选标记等。表达载体都是 穿梭质粒，先在大肠杆菌复制扩 增，然后被导入宿主酵母细胞。 为使产物分泌胞外，表达载体还 需带有信号肽序列。
生产过程
（1）细胞增殖繁衍；
（2）分批流加的过渡阶段（甘油或葡萄糖）； （3）诱导表达阶段（甲醇）；
各阶段碳源都为限制 性基质,其补加速率的 动力学模型是高效表 达的基础。
甲型优点
(1)具有醇氧化酶AOX1基因启动子，这是目前最强，调
控机理最严格的启动子之一；
(2)表达效率高,其表达的外源蛋白可占总表达蛋白的 90%以上,有利于目的蛋白的分离纯化； (3)在简单合成培养基中可实现高密度培养； (4)表达质粒能在基因组的特定位点以单拷贝或多拷贝 的形式稳定整合； (5)由于该酵母可以以甲醇为唯一碳源和能源,而绝大多 数微生物并不能以甲醇为碳源,可以减少污染。Fra bibliotek甲型不足
(1)发酵周期长；
(2)甲醇易燃易爆有毒，存在一 定的危险性； (3)筛选高产菌株需用的药物价 格比较昂贵； (4)培养基和培养条件不成熟。
毕赤酵母表达手册
Pichia Expression KitVersion M01110225-0043Pichia Expression KitA Manual of Methods for Expression of Recombinant Proteins in Pichia pastorisCatalog no. K1710-01tech_service@iiINDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTThe Pichia Expression Kit is based on the yeast Pichia pastoris. Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology/Industry Associates (SIBIA) for high-level expression of recombinant proteins. All patents for Pichia pastoris and licenses for its use as an expression system are owned by Research Corporation Technologies, Inc. Tucson, Arizona. Invitrogen has an exclusive license to sell the Pichia Expression Kit to scientists for research purposes only, under the terms described below. Use of Pichia pastoris by commercial corporations requires the user to obtain a commercial license as detailed below. Before using the Pichia Expression Kit, please read the following license a greement. If you do not agree to be bound by its terms, contact Invitrogen within 10 days for authorization to return the unused Pichia Expression Kit and to receive a full credit. If you do agree to the terms of this Agreement, please complete the User Registration Card and return it to Invitrogen before using the kit.INDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTInvitrogen Corporation (INVITROGEN) grants you a non-exclusive license to use the enclosed Pichia Expression Kit (EXPRESSION KIT) for academic research or for evaluation purposes only. The EXPRESSION KIT is being transferred to you in furtherance of, and reliance on, such license. You may not use the EXPRESSION KIT, or the materials contained therein, for any commercial purpose without a license for such purpose from RESEARCH CORPORATION TECHNOLOGIES, INC., Tucson, Arizona. Commercial purposes include the use in or sale of expressed proteins as a commercial product, or use to facilitate or advance research or development of a commercial product. Commercial entities may conduct their evaluation for one year at which time this license automatically terminates. Commercial entities will be contacted by Research Corporation Technologies during the evaluation period regarding the purchase of a commercial license.Access to the EXPRESSION KIT must be limited solely to those officers, employees and students of your institution who need access thereto in order to perform the above-described research or evaluation. You must inform each of such officer, employee and student of the provisions of this Agreement and require them to agree, in writing, to be bound by the provisions of this Agreement. You may not distribute the EXPRESSION KIT to others, even those within your own institution. You may transfer modified, altered or original material from the EXPRESSION KIT to a third party following notification of INVITROGEN such that the recipient can be licensed. You may not assign, sub-license, rent lease or otherwise transfer this License or any of the rights or obligation hereunder, except as expressly permitted.This License is effective until terminated. You may terminate it at any time by destroying all Pichia expression products in your control. It will also terminate automatically if you fail to comply with the terms and conditions of the Agreement. You shall, upon termination of the License, destroy all Pichia Expression Kits in your control, and so notify INVITROGEN in writing.This License Shall be governed in its interpretation and enforcement by the laws of the State of California.Product User Registration CardPlease complete and return the enclosed Product User Registration Card for each Pichia Expression Kit that you purchase. This will serve as a record of your purchase and registration and will allow Invitrogen to provide you with technical support and manual updates. It will also allow Invitrogen to update you on future developments of and improvements to the Pichia Expression Kit. The agreement outlined above becomes effective upon our receipt of your User Registration Card or 10 days following the sale of the Pichia Expression Kit to you. Use of the kit at any time results in immediate obligation to the terms and conditions stated in this Agreement.Technical ServicesInvitrogen provides Technical Services to all of our registered Pichia Expression Kit users. Please contact us if you need assistance with the Pichia Expression Kit.United States Headquarters:Japanese Headquarters European Headquarters:Invitrogen Corporation1600 Faraday AvenueCarlsbad, CA 92008 USATel: 1 760 603 7200Tel (Toll Free): 1 800 955 6288 Fax: 1 760 602 6500E-mail:tech_service@ Invitrogen Japan K.K.Nihonbashi Hama-Cho Park Bldg. 4F2-35-4, Hama-Cho, NihonbashiTel: 81 3 3663 7972Fax: 81 3 3663 8242E-mail: jpinfo@Invitrogen Ltd3 Fountain DriveInchinnan Business ParkPaisley PA4 9RF, UKTel (Free Phone Orders): 0800 269 210Tel (General Enquiries): 0800 5345 5345Fax: +44 (0) 141 814 6287E-mail: eurotech@iiiivTable of ContentsMaterials (vii)Purchaser Notification (x)Product Qualification (xii)Introduction (1)Overview (1)Experimental Outline (3)Recombination and Integration in Pichia (7)Methods (11)Pichia Strains (11)E. coli Strains (13)Selecting a Pichia Expression Vector (14)pHIL-D2 (16)pPIC3.5 (17)pHIL-S1 (18)pPIC9 (19)Signal Sequence Processing (20)Cloning into the Pichia Expression Vectors (21)Transformation into E. coli (26)Preparation of Transforming DNA (27)Growth of Pichia for Spheroplasting (30)Preparation of Spheroplasts (32)Transformation of Pichia (34)Screening for Mut+ and Mut S Transformants (36)PCR Analysis of Pichia Integrants (40)Expression of Recombinant Pichia Strains (42)Analysis by SDS-Polyacrylamide Gel Electrophoresis (45)Optimization of Pichia Protein Expression (47)Scale-up of Expression (49)Protein Purification and Glycosylation (51)Recipes (53)E. coli Media Recipes (53)Pichia Media Recipes (54)Appendix (59)Electroporation of Pichia (59)PEG 1000 Transformation Method for Pichia (60)Lithium Chloride Transformation Method (61)Total DNA Isolation from Pichia (62)Detection of Multiple Integration Events (63)Procedure for Total RNA Isolation from Pichia (64)β-Galactosidase Assay (65)Technical Service (67)References (69)vviMaterialsKit Contents Box 1: Spheroplast Module. Store at room temperature.Reagent Amount ComponentsSOS media 20 ml 1 M Sorbitol0.3X YPD10 mM CaCl2Sterile Water 2 x 125 ml Autoclaved, deionized waterSE 2 x 125 ml 1 M Sorbitol25 mM EDTA, pH 8.0SCE 2 x 125 ml 1 M Sorbitol10 mM Sodium citrate buffer, pH 5.81 mM EDTA1 M Sorbitol2 x 125 ml --CaS 2 x 60 ml 1 M Sorbitol10 mM Tris-HCl, pH 7.5;10 mM CaCl240% PEG 25 ml 40% (w/v) PEG 3350 (Reagent grade) in waterCaT 25 ml 20 mM Tris-HCl, pH 7.520 mM CaCl2Stab Vials: Pichia and E. coli stabs. Store at +4°C.Phenotype(Pichia only)GenotypeStrain Amountstab his4Mut+GS115 1stab arg4 his4 aox1::ARG4 Mut S, Arg+KM71 1GS115 Albumin 1 stab HIS4Mut SGS115 β-Gal 1 stab HIS4Mut+stab F´ {pro AB, lac I q, lac Z∆M15, Tn10 (Tet R)} mcr A,TOP10F´ 1∆(mrr-hsd RMS-mcr BC), φ80lac Z∆M15, ∆lac X74,deo R, rec A1, ara D139, ∆(ara-leu)7697, gal U,gal K, rps L (Str R), end A1, nup G λ-.Box 2: Spheroplast Module. Store at -20°C.ComponentsReagent AmountZymolyase 10 x 20 µl 3 mg/ml Zymolyase in water(100,000 units/g lytic activity)1 M DTT 10 x 1 ml 1 M dithiothreitol in watercontinued on next pageviiKit Contents,continuedVector Box. Store at -20°C.Reagent DescriptionpHIL-D210 µg, lyophilized in TE, pH 8.0Vector for intracellular expression in PichiapPIC3.510 µg, lyophilized in TE, pH 8.0Vector for intracellular expression in PichiapHIL-S110 µg, lyophilized in TE, pH 8.0 Vector for secreted expression in Pichia. Uses the PHO1 signal sequencepPIC910 µg, lyophilized in TE, pH 8.0 Vector for secreted expression in Pichia. Uses the α-factor signal sequencePrimer Box. Store at -20°C.5´ AOX1 sequencing primer2 µg (312 pmoles), lyophilized5´-GACTGGTTCCAATTGACAAGC-3´3´ AOX1 sequencing primer2 µg (314 pmoles), lyophilized5´-GCAAATGGCATTCTGACATCC-3´α-Factor sequencing primer2 µg (315 pmoles), lyophilized5´-TACTATTGCCAGCATTGCTGC-3´Media The following prepackaged media is included for your convenience. Instructions for use are provided on the package.Media Amount Yield YP Base Medium 2 pouches 2 liters of YP mediumYP Base Agar Medium 2 pouches 2 liters of YP mediumYeast Nitrogen Base 1 pouch 500 ml of 10X YNBFor transformation of Pichia by spheroplasting, the Pichia Spheroplast Module isavailable separately from Invitrogen (see below for ordering information).Product Reactions or Amount Catalog no.Pichia Spheroplast Module 10 spheroplast preparations(50 transformations)K1720-01continued on next pageviiiRequired Equip-ment and Supplies (not provided) • 30°C rotary shaking incubator• Water baths capable of 37°C, 45°C, and 100°C• Centrifuge suitable for 50 ml conical tubes (floor or table-top)• Baffled culture flasks with metal covers (50 ml, 250 ml, 500 ml, 1000 ml, and 3 L)• 50 ml sterile, conical tubes• 6 ml and 15 ml sterile snap-top tubes (Falcon 2059 or similar)• UVSpectrophotometer• Mini agarose gel apparatus and buffers• Polyacrylamide Gel Electrophoresis apparatus and buffers• Media for transformation, growth, screening, and expression (see Recipes, pages 53-58) • 5% SDS solution (10 ml per transformation)• Sterile cheesecloth or gauze• Breaking Buffer (see Recipes, page 58)• Acid-washed glass beads (available from Sigma)• Replica-plating equipment (optional)• BeadBreaker™ (optional)ixPurchaser NotificationIntroduction The Pichia Expression Kit is based on the yeast Pichia pastoris. Pichia pastoris wasdeveloped into an expression system by scientists at Salk Institute Biotechnology/ IndustryAssociates (SIBIA) and Phillips Petroleum for high-level expression of recombinantproteins. All patents for Pichia pastoris and licenses for its use as an expression system areowned by Research Corporation Technologies (RCT), Inc., Tucson, Arizona. Forinformation on commercial licenses, please see page x.The Nature of the Invitrogen License Invitrogen has an exclusive license to sell the Pichia Expression Kit to scientists for research purposes only, under the terms described below. Use of Pichia pastoris by commercial entities for any commercial purpose requires the user to obtain a commercial license as detailed below. Before using the Pichia Expression Kit, please read the following license agreement. If you do not agree to be bound by its terms, contact Invitrogen within 10 days for authorization to return the unused Pichia Expression Kit and to receive a full credit. If you do agree to the terms of this license agreement, please complete the User Registration Card and return it to Invitrogen before using the kit.Pichia pastoris Patents Pichia pastoris is covered by one or more of the following U.S. patents and corresponding foreign patents owned and licensed by Research Corporation Technologies:4,683,293 4,808,537 4,812,405 4,818,700 4,837,148 4,855,231 4,857,467 4,879,231 4,882,279 4,885,242 4,895,800 4,929,555 5,002,876 5,004,688 5,032,516 5,122,465 5,135,868 5,166,329Individual Pichia Expression Kit License Agreement Invitrogen Corporation ("Invitrogen") grants you a non-exclusive license to use the enclosed Pichia Expression Kit ("Expression Kit") for academic research or for evaluation purposes only. The Expression Kit is being transferred to you in furtherance of, and reliance on, such license. You may not use the Expression Kit, or the materials contained therein, for any commercial purpose without a license for such purpose from Research Corporation Technologies, Inc., Tucson, Arizona.Definition of Commercial Purpose Commercial purposes include:(a) any use of Expression Products in a Commercial Product(b) any use of Expression Products in the manufacture of a Commercial Product(c) any sale of Expression Products(d) any use of Expression Products or the Expression Kit to facilitate or advanceresearch or development of a Commercial Product(e) any use of Expression Products or the Expression Kit to facilitate or advance anyresearch or development program the results of which will be applied to thedevelopment of Commercial Products"Expression Products" means products expressed with the Expression Kit, or with the use of any vectors or host strains in the Expression Kit. "Commercial Product" means any product intended for sale or commercial use.Commercial entities may conduct their evaluation for one year at which time this license automatically terminates. Research Corporation Technologies will contact commercial entities during the evaluation period regarding their desire for a commercial license.continued on next pagexPurchaser Notification, continuedIndividual Responsibilities Access to the Expression Kit must be limited solely to those officers, employees and students of your institution who need access to perform the above-described research or evaluation. You must inform each such officer, employee and student of the provisions of this license agreement and require them to agree, in writing, to be bound by the provisions of this license agreement. You may not distribute neither the Expression Kit nor the vectors or host strains contained in it to others, even to those within your own institution. You may only transfer modified, altered, or original material from the Expression Kit to a third party following written notification of, and written approval from, Invitrogen so that the recipient can be licensed. You may not assign, sub-license, rent, lease or otherwise transfer this license agreement or any of the rights or obligation thereunder, except as expressly permitted by Invitrogen and RCT.Termination of License This license agreement is effective until terminated. You may terminate it at any time by destroying all Pichia expression products in your control. It will also terminate auto-matically if you fail to comply with the terms and conditions of the license agreement. You shall, upon termination of the license agreement, destroy all Pichia Expression Kits in your control, and so notify Invitrogen in writing.This License shall be governed in its interpretation and enforcement by the laws of the State of California.Contact for Commercial Licensing Bennett Cohen, Ph.D.Research Corporation Technologies 101 North Wilmot Road, Suite 600 Tucson, Arizona 85711-3335 Phone: (520) 748-4400Fax: (520)748-0025User Registration Card Please complete and return the enclosed User Registration Card for each PichiaExpression Kit that you purchase. This will serve as a record of your purchase and regis-tration and will allow Invitrogen to provide you with technical support and manualupdates. It will also allow Invitrogen to update you on future developments and improve-ments to the Pichia Expression Kit. The agreement outlined above becomes effectiveupon our receipt of your User Registration Card or 10 days following the sale of thePichia Expression Kit to you. Use of the kit at any time results in immediate obligation tothe terms and conditions stated in this license agreement.xiProduct QualificationIntroduction This section describes the criteria used to qualify the components in the PichiaExpression Kit.Vectors All expression vectors are qualified by restriction enzyme digestion. Restriction digests must demonstrate the correct banding pattern when electrophoresed on an agarose gel.Spheroplast Reagents The spheroplast reagents are qualified by spheroplast preparation of GS115 following the protocol provided in the Pichia Expression Kit manual. At least 70% of the Pichia pastoris cells must form spheroplasts in 30 minutes or less.Pichia Strains The Pichia strains are by demonstrating viability of the culture. Single colonies should arise within 48 hours after streaking on YPD medium from the stabPrimers Sequencing primers are lot tested by automated DNA sequencing experiments.Buffers andSolutionsAll buffers and solutions are extensively tested for sterility.Media All Pichia growth and expression media are qualified by growing the GS115 Pichiastrain.xiiIntroductionOverviewReview Articles The information presented here is designed to give you a concise overview of the Pichia pastoris expression system. It is by no means exhaustive. For further information, pleaseread the articles cited in the text along with recent review articles (Buckholz and Gleeson,1991; Cregg et al., 1993; Sreekrishna et al., 1988; Wegner, 1990). A general review offoreign gene expression in yeast is also available (Romanos et al., 1992).General Characteristics of Pichia pastoris As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression levels. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces, and has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia very useful as a protein expression system.Similarity to Saccharomyces Many of the techniques developed for Saccharomyces may be applied to Pichia including: • transformation by complementation• genedisruption• genereplacementIn addition, the genetic nomenclature used for Saccharomyces has been applied to Pichia. For example, the HIS4 gene in both Saccharomyces and Pichia encodes histidinol dehydrogenase. There is also cross-complementation between gene products in both Saccharomyces and Pichia. Several wild-type genes from Saccharomyces complement comparable mutant genes in Pichia. Genes such as HIS4, LEU2, ARG4, TRP1, and URA3 all complement their respective mutant genes in Pichia.Pichia pastoris as a Methylotrophic Yeast Pichia pastoris is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen by the enzyme alcohol oxidase. This reaction generates both formaldehyde and hydrogen peroxide. To avoid hydrogen peroxide toxicity, methanol metabolism takes place within a specialized cell organelle called the peroxisome, which sequesters toxic by-products from the rest of the cell. Alcohol oxidase has a poor affinity for O2, and Pichia pastoris compensates by generating large amounts of the enzyme. The promoter regulating the production of alcohol oxidase drives heterologous protein expression in Pichia.Two Alcohol Oxidase Proteins The AOX1 and AOX2 genes code for alcohol oxidase in Pichia pastoris. The AOX1 gene product accounts for the majority of alcohol oxidase activity in the cell. Expression of the AOX1 gene is tightly regulated and induced by methanol to high levels, typically > 30% ofthe total soluble protein in cells grown with methanol as the carbon source. The AOX1 gene has been isolated and the AOX1 promoter is used to drive expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a). While AOX2 is about 97% homologous to AOX1, growth on methanol is much slower than with AOX1. This slowgrowth allows isolation of Mut S strains (aox1) (Cregg et al., 1989; Koutz et al., 1989).continued on next page1Overview, continuedExpression Expression of the AOX1 gene is controlled at the level of transcription. In methanol-grown cells approximately 5% of the polyA+ RNA is from the AOX1 gene. The regulation of theAOX1 gene is a two step process: a repression/derepression mechanism plus an inductionmechanism (e.g. GAL1 gene in Saccharomyces (Johnston, 1987)). Briefly, growth onglucose represses transcription, even in the presence of the inducer methanol. For thisreason, growth on glycerol is recommended for optimal induction with methanol. Pleasenote that growth on glycerol (derepression) is not sufficient to generate even minute levelsof expression from the AOX1 gene. The inducer, methanol, is necessary for detectablelevels of AOX1 expression (Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a).Phenotype of aox1 mutants Loss of the AOX1 gene, and thus a loss of most of the cell's alcohol oxidase activity, results in a strain that is phenotypically Mut S (Methanol utilization slow). This has in the past been referred to as Mut. The Mut S designation has been chosen to accurately describe the phenotype of these mutants. This results in a reduction in the cells' ability to metabolize methanol. The cells, therefore, exhibit poor growth on methanol medium. Mut+ (Methanol utilization plus) refers to the wild type ability of strains to metabolize methanol as the sole carbon source. These two phenotypes are used when evaluating Pichia transformants for integration of your gene (Experimental Outline, page 3).Intracellular and Secretory Protein Expression Heterologous expression in Pichia can be either intracellular or secreted. Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. While several different secretion signal sequences have been used successfully, including the native secretion signal present on some heterologous proteins, success has been variable. The secretion signal sequence from the Saccharomyces cerevisiaeα factor prepro peptide has been used most successfully (Cregg et al., 1993; Scorer et al., 1993).The major advantage of expressing heterologous proteins as secreted proteins is that Pichia pastoris secretes very low levels of native proteins. That, combined with the very low amount of protein in the minimal Pichia growth medium, means that the secreted heterologous protein comprises the vast majority of the total protein in the medium and serves as the first step in purification of the protein (Barr et al., 1992). Note: If there are recognized glycosylation sites (Asn-X-Ser/Thr) in your protein's primary sequence, glycosylation may occur at these sites.Posttranslational Modifications In comparison to Saccharomyces cerevisiae, Pichia may have an advantage in the glyco-sylation of secreted proteins because it may not hyperglycosylate. Both Saccharomyces cerevisiae and Pichia pastoris have a majority of N-linked glycosylation of the high-mannose type; however, the length of the oligosaccharide chains added posttranslationally to proteins in Pichia (average 8-14 mannose residues per side chain) is much shorter than those in S. cerevisiae (50-150 mannose residues) (Grinna and Tschopp, 1989; Tschopp et al., 1987b). Very little O-linked glycosylation has been observed in Pichia.In addition, Saccharomyces cerevisiae core oligosaccharides have terminal α1,3 glycan linkages whereas Pichia pastoris does not. It is believed that the α1,3 glycan linkages in glycosylated proteins produced from Saccharomyces cerevisiae are primarily responsible for the hyper-antigenic nature of these proteins making them particularly unsuitable for therapeutic use. Although not proven, this is predicted to be less of a problem for glycoproteins generated in Pichia pastoris, because it may resemble the glycoprotein structure of higher eukaryotes (Cregg et al., 1993).2Experimental OutlineSelection of Vector and Cloning To utilize the strong, highly inducible P AOX1 promoter for expression of your protein, four expression vectors are included in this kit. pHIL-D2 and pPIC3.5 are used for intracellular expression while pHIL-S1 and pPIC9 are used for secreted expression (see pages 14-19 for more information). Before cloning your insert, you must...• decide whether you want intracellular or secreted expression.• analyze your insert for the following restriction sites: Sac I, Stu I, Sal I, Not I, and Bgl II. These sites are recommended for linearizing your construct prior to Pichiatransformation. If your insert has all of these sites, see pages 28-29 for alternate sites.Transformation and IntegrationTwo different phenotypic classes of His+ recombinant strains can be generated: Mut+ and Mut S. Mut S refers to the "Methanol utilization slow" phenotype caused by the loss of alcohol oxidase activity encoded by the AOX1 gene. A strain with a Mut S phenotype has a mutant aox1 locus, but is wild type for AOX2. This results in a slow growth phenotype on methanol medium. Transformation of strain GS115 can yield both classes of transformants, His+ Mut+ and His+Mut S, while KM71 yields only His+ Mut S since the strain itself is Mut S. Both Mut+ and Mut S recombinants are useful to have as one phenotype may favor better expression of your protein than the other. Due to clonal variation, you should test 6-10 recombinants per phenotype. There is no way to predict beforehand which construct or isolate will better express your protein. We strongly recommend that you analyze Pichia recombinants by PCR to confirm integration of your construct (see page 40).Once you have successfully cloned your gene, you will then linearize your plasmid to stimulate recombination when the plasmid is transformed into Pichia. The table below describes the types of recombinants you will get by selective digestion of your plasmid. RestrictionEnzymeIntegration Event GS115 Phenotype KM71 PhenotypeSal I or Stu I Insertion at his4His+ Mut+ His+ Mut SSac I Insertion at 5´AOX1 regionHis+ Mut+ His+ Mut SNot I or Bgl II Replacement atAOX1 locusHis+ Mut SHis+ Mut+His+ Mut S (notrecommended, see page 11)Expression and Scale-up After confirming your Pichia recombinants by PCR, you will test expression of both His+Mut+ and His+ Mut S recombinants. This will involve growing a small culture of each recombinant, inducing with methanol, and taking time points. If looking for intracellular expression, analyze the cell pellet from each time point by SDS polyacrylamide gel electrophoresis (SDS-PAGE). If looking for secreted expression, analyze both the cellpellet and supernatant from each time point. We recommend that you analyze your SDS-PAGE gels by both Coomassie staining and Western blot, if you have an antibody to your protein. We also suggest checking for protein activity by assay, if one is available. Not all proteins express to the level of grams per liter, so it is advisable to check by Western blotor activity assay, and not just by Coomassie staining of SDS-PAGE gels for production of your protein.Choose the Pichia recombinant strain that best expresses your protein and optimizeinduction based on the suggestions on pages 47-48. Once expression is optimized, scale-up your expression protocol to produce more protein.continued on next page3。
毕赤酵母的培养基1.
毕赤酵母的培养巴斯德毕赤酵母是甲醇营养型酵母中的一类能够利用甲醇作为唯一碳源和能源的酵母菌。
与其它酵母一样，在无性生长期主要以单倍体形式存在，当环境营养限制时，常诱导2个生理类型不同的接合型单倍体细胞交配，融合成双倍体。
比赤酵母表达常用的培养基：1 LB培养基胰蛋白胨1% 酵母粉0.5% 氯化钠1% PH 7.0----------------大肠杆菌培养制作平板加入2%的琼脂粉，121摄氏度20分钟，可于室温保存。
用于培养pPICZ αA 原核宿主菌TOP10F’时可加入Zeocin 25ug / mL（博来霉素，抗菌素）宿主细胞His- -----------诱变造成的。
MD------------酵母转化筛选培养基（营养缺陷型His-）13.4g/L酵母基本氮源；0.4mg/L生物素；20g/L葡萄糖用于表达的毕赤酵母都属于组氨酸缺陷型的只有染色体上成功整合入重组质粒载体基因的毕赤酵母菌株才能在组氨酸缺失的MD培养基生长，以此筛选出重组子。
MM--------------酵母转化进一步筛选培养基仅能满足微生物野生型菌株生长需要的培养基，成为基本培养基（minimal medium,MM），有时用符号“[ －]”来表示。
不同微生物的基本培养基是不相同的。
2 LLB 培养基胰蛋白胨1% 酵母粉0.5% 氯化钠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pPICZ αA 原核宿主菌TOP10F’时，加入Zeocin 25ug / ml，可以4℃条件下保存1～2 周.3 YPD培养基酵母粉1%，胰蛋白胨2%，葡萄糖2%，固体加2%的琼脂粉，YPD 培养基可常温保存,是毕赤酵母的最基本培养基，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。
4 BMGY培养基酵母粉1% 蛋白胨2%，磷酸钾缓冲溶液平PH 6 100mmol/L YNB（酵母氮源）1.34% 生物素（4×10 -5）%甘油1%，毕赤酵母诱导表达前培养基，YNB和Biotin过滤除菌。
毕赤酵母蛋白质组学研究课件PPT
There is no significant decrease in the concentration of HBsAg even after
prolonged cultivation suggesting that the HBsAg deposits in the ER are
we20ll21p/3ro/1t0ected from proteolytic degradation.
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蛋 白 质 组 学 技 术 路 线
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内容概要
➢蛋白质组学技术介绍 ➢毕赤酵母蛋白质组学研究进展
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2.1 毕赤酵母的基因组
菌株
GS115 DSMZ 70382
CBS 7435
基因数（个）
5313 5450 5007
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2.2 毕赤酵母的蛋白质组学研究概况
21
13
2.2.4 X-33——经甲醇诱导高表达胰岛素前体的响应
2 g/L, 48 hrs
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UPR及ERAD相关蛋白下调表达
UPR 通过相关感受器发出一系列的调节信号用: 提高内质网的折叠能力; 加快错误折叠或未折叠蛋白的降解; 限制 mRNA 翻译速度;
以减轻客户蛋白负荷。
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2.2.2 渗透压对毕赤酵母表达系统的影响
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2.2.3 GS 115——经甲醇诱导高表达HBsAg的响应
6 g/L,114 hrs
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碳代谢通路
甲醇异化代谢相关蛋 白上调表达；
毕赤酵母的培养基1
毕赤酵母的培养巴斯德毕赤酵母是甲醇营养型酵母中的一类能够利用甲醇作为唯一碳源和能源的酵母菌。
与其它酵母一样，在无性生长期主要以单倍体形式存在，当环境营养限制时，常诱导2个生理类型不同的接合型单倍体细胞交配，融合成双倍体。
比赤酵母表达常用的培养基：1 LB培养基胰蛋白胨1% 酵母粉0.5% 氯化钠1% PH 7.0----------------大肠杆菌培养制作平板加入2%的琼脂粉，121摄氏度20分钟，可于室温保存。
用于培养pPICZ αA 原核宿主菌TOP10F’时可加入Zeocin 25ug / mL（博来霉素，抗菌素）宿主细胞His- -----------诱变造成的。
MD------------酵母转化筛选培养基（营养缺陷型His-）13.4g/L酵母基本氮源；0.4mg/L生物素；20g/L葡萄糖用于表达的毕赤酵母都属于组氨酸缺陷型的只有染色体上成功整合入重组质粒载体基因的毕赤酵母菌株才能在组氨酸缺失的MD培养基生长，以此筛选出重组子。
MM--------------酵母转化进一步筛选培养基仅能满足微生物野生型菌株生长需要的培养基，成为基本培养基（minimal medium,MM），有时用符号“[ －]”来表示。
不同微生物的基本培养基是不相同的。
2 LLB 培养基胰蛋白胨1% 酵母粉0.5% 氯化钠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pPICZ αA 原核宿主菌TOP10F’时，加入Zeocin 25ug / ml，可以4℃条件下保存1～2 周.3 YPD培养基酵母粉1%，胰蛋白胨2%，葡萄糖2%，固体加2%的琼脂粉，YPD 培养基可常温保存,是毕赤酵母的最基本培养基，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。
4 BMGY培养基酵母粉1% 蛋白胨2%，磷酸钾缓冲溶液平PH 6 100mmol/L YNB（酵母氮源）1.34% 生物素（4×10 -5）%甘油1%，毕赤酵母诱导表达前培养基，YNB和Biotin过滤除菌。