毕赤酵母表达操作手册(精译版)

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毕赤酵母翻译

Glycoengineered(糖基化工程) 毕赤 pastoris(毕赤酵母) 中表达的重组单克隆抗体的纯化工艺开发摘要一个强健的和可扩展的净化过程被开发快速从 glycoengineered 毕赤酵母发酵生成抗体的纯度高、数量充足。

蛋白 A 亲和层析用于捕获从发酵液抗体。

PH 梯度洗脱已应用到要防止抗体沉淀在低 pH 的蛋白 A 列。

从蛋白抗体色谱法所载一些产品的相关杂质，被劈重链、重链和轻链的 misassembling。

它也有一些处理相关的杂质，包括蛋白 A 残留、 endotoxin(内毒素)、宿主细胞 DNA 和蛋白质。

Ph 值为 4.5-6.0 的最优氯化钠渐变阳离子交换色谱法有效去除这些产品和过程相关的杂质。

从 glycoengineered P.酵母抗体是其 heterotetramer 折叠、物理稳定性和约束力的亲和性的商业同行相媲美。

介绍单克隆抗体（Mab）正迅速成为关键产品的生物医药产业。

大多数商业治疗性抗体是原封 IgG 分子与 IgG1、 IgG2 正在共同的子类。

这些 IgGs 由调解抗原和效应器函数之间的联系，在免疫过程中发挥中心作用。

对目标细胞的特异性抗原的 igg 抗体可变域的绑定将抗体依赖的细胞毒作用 (ADCC) 及补体依赖性细胞毒性 (CDC) 定向杀害的目标单元格。

ADCC 触发由交联体 Fc 域的 IgG 抗体（Fc(Rs)，尤其是 Fc (RI 和Fc (制证机对免疫效应细胞。

可能调解 ADCC 效应细胞包括自然杀手细胞、巨噬细胞和嗜中性粒细胞。

疾病预防控制中心是由补充组件 C1q 绑定到 IgG，触发蛋白水解的级联，以激活补充 fc 功能区启动。

IgG 型抗体有两重链和两条轻链由分子内的二硫键形成 heterotetramer 在一起。

重链是通过共价键固定在天冬酰胺 297 (Asn-297) 低聚糖的糖基化。

人免疫球蛋白的主要N-聚糖复杂 biantennary (二分支的) 类型，有 '核心' heptasaccharide(七庚糖)，GlcNac2Man3GlcNac2，被称为 G0 在我们的工作和文学。

毕赤酵母手册

毕赤酵母表达实验手册作者：Jnuxz 来源：丁香园时间：2007-9-5大肠杆菌表达系统最突出的优点是工艺简单、产量高、周期短、生产成本低。

然而，许多蛋白质在翻译后，需经过翻译后的修饰加工，如磷酸化、糖基化、酰胺化及蛋白酶水解等过程才能转化成活性形式。

大肠杆菌缺少上述加工机制，不适合用于表达结构复杂的蛋白质。

另外，蛋白质的活性还依赖于形成正确的二硫键并折叠成高级结构，在大肠杆菌中表达的蛋白质往往不能进行正确的折叠，是以包含体状态存在。

包含体的形成虽然简化了产物的纯化，但不利于产物的活性，为了得到有活性的蛋白，就需要进行变性溶解及复性等操作，这一过程比较繁琐，同时增加了成本。

大肠杆菌是用得最多、研究最成熟的基因工程表达系统，当前已商业化的基因工程产品大多是通过大肠杆菌表达的，其主要优点是成本低、产量高、易于操作。

但大肠杆菌是原核生物，不具有真核生物的基因表达调控机制和蛋白质的加工修饰能力，其产物往住形成没有活性的包涵体，需要经过变性、复性等处理，才能应用。

近年来，以酵母作为工程菌表达外源蛋白日益引起重视，原因是与大肠杆菌相比，酵母是低等真核生物，除了具有细胞生长快，易于培养，遗传操作简单等原核生物的特点外，又具有真核生物时表达的蛋白质进行正确加工，修饰，合理的空间折叠等功能，非常有利于真核基因的表达，能有效克服大肠杆菌系统缺乏蛋白翻译后加工、修饰的不足。

因此酵母表达系统受到越来越多的重视和利用。

［1］。

同时与大肠杆菌相比，作为单细胞真核生物的酵母菌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。

酿酒酵母（Saccharomyces.Cerevisiae）在分子遗传学方面被人们的认识最早，也是最先作为外源基因表达的酵母宿主。

1981年酿酒酵母表达了第一个外源基因----干扰素基因［2］，随后又有一系列外源基因在该系统得到表达［3、4、5、6］。

干扰素和胰岛素虽然已经利用酿酒酵母大量生产并被广泛应用，当利用酿酒酵母制备时，实验室的结果很令人鼓舞，但由实验室扩展到工业规模时，其产量迅速下降。

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。

毕赤酵母发酵手册

Version B Pichia Fermentation Process GuidelinesOverviewIntroduction Pichia pastoris, like Saccharomyces cerevisiae, is particularly well-suited forfermentative growth. Pichia has the ability to reach very high cell densities duringfermentation which may improve overall protein yields.We recommend that only those with fermentation experience or those who have accessto people with experience attempt fermentation. Since there are a wide variety offermenters available, it is difficult to provide exact procedures for your particular case.The guidelines given below are based on fermentations of both Mut+ and Mut S Pichiastrains in a 15 liter table-top glass fermenter. Please read the operator's manual for yourparticular fermenter before beginning. The table below provides an overview of thematerial covered in these guidelines.Step Topic Page1 Fermentationparameters 12 Equipment needed and preparation of medium 23 Measurement and use of dissolved oxygen (DO) in the culture 34 Growth of the inoculum 45 Generation of biomass on glycerol in batch and fed-batch phases 4-56 Induction of expression of Mut+ and Mut S recombinants in themethanol fed-batch phase6-77 Harvesting and lysis of cells 88 References 9-109 Recipes 11Fermentation Parameters It is important to monitor and control the following parameters throughout thefermentation process. The following table describes the parameters and the reasons for monitoring them.Parameter Reason Temperature (30.0°C) Growth above 32°C is detrimental to protein expression Dissolved oxygen (>20%) Pichia needs oxygen to metabolize glycerol andmethanolpH (5.0-6.0 and 3.0) Important when secreting protein into the medium andfor optimal growthAgitation (500 to 1500 rpm) Maximizes oxygen concentration in the mediumAeration (0.1 to 1.0 vvm*for glass fermenters)Maximizes oxygen concentration in the medium whichdepends on the vesselAntifoam (the minimumneeded to eliminate foam)Excess foam may cause denaturation of your secretedprotein and it also reduces headspaceCarbon source (variablerate)Must be able to add different carbon sources at differentrates during the course of fermentationcontinued on next pageOverview, continuedRecommended Equipment Below is a checklist for equipment recommendations.• A jacketed vessel is needed for cooling the yeast during fermentation, especially during methanol induction. You will need a constant source of cold water (5-10°C). This requirement may mean that you need a refrigeration unit to keep the water cold. • A foam probe is highly recommended as antifoam is required.• A source of O2--either air (stainless steel fermenters at 1-2 vvm) or pure O2(0.1-0.3 vvm for glass fermenters).• Calibrated peristaltic pumps to feed the glycerol and methanol.• Automatic control of pH.Medium Preparation You will need to prepare the appropriate amount of following solutions:• Fermentation Basal Salts (page 11)• PTM1Trace Salts (page 11)• ~75 ml per liter initial fermentation volume of 50% glycerol containing 12 ml PTM1 Trace Salts per liter of glycerol.• ~740 ml per liter initial fermentation volume of 100% methanol containing 12 mlPTM1Trace Salts per liter of methanol.Monitoring the Growth of Pichia pastoris Cell growth is monitored at various time points by using the absorbance at 600 nm (OD600) and the wet cell weight. The metabolic rate of the culture is monitored by observing changes in the concentration of dissolved oxygen in response to carbon availability (see next page).Dissolved Oxygen (DO) MeasurementIntroduction The dissolved oxygen concentration is the relative percent of oxygen in the mediumwhere 100% is O2-saturated medium. Pichia will consume oxygen as it grows, reducing the dissolved oxygen content. However, because oxygen is required for the first step ofmethanol catabolism, it is important to maintain the dissolved oxygen (DO) concentra-tion at a certain level (>20%) to ensure growth of Pichia on methanol. Accuratemeasurement and observation of the dissolved oxygen concentration of a culture willgive you important information about the state and health of the culture. Therefore, it isimportant to accurately calibrate your equipment. Please refer to your operator's manual.Maintaining the Dissolved Oxygen Concentration (DO) 1. Maintaining the dissolved oxygen above 20% may be difficult depending on theoxygen transfer rates (OTR) of the fermenter, especially in small-scale glassvessels. In a glass vessel, oxygen is needed to keep the DO above 20%, usually~0.1-0.3 vvm (liters of O2per liter of fermentation culture per minute). Oxygen consumption varies and depends on the amount of methanol added and the protein being expressed.2. Oxygen can be used at 0.1 to 0.3 vvm to achieve adequate levels. This can beaccomplished by mixing with the air feed and can be done in any glass fermenter.For stainless steel vessels, pressure can be used to increase the OTR. Be sure toread the operator's manual for your particular fermenter.3. If a fermenter cannot supply the necessary levels of oxygen, then the methanol feedshould be scaled back accordingly. Note that decreasing the amount of methanol may reduce the level of protein expression.4. To reach maximum expression levels, the fermentation time can be increased todeliver similar levels of methanol at the lower feed rate. For many recombinantproteins, a direct correlation between amount of methanol consumed and theamount of protein produced has been observed.Use of DO Measurements During growth, the culture consumes oxygen, keeping the DO concentration low. Note that oxygen is consumed whether the culture is grown on glycerol or methanol. The DO concentration can be manipulated to evaluate the metabolic rate of the culture and whether the carbon source is limiting. The metabolic rate indicates how healthy the culture is. Determining whether the carbon source is limiting is important if you wish to fully induce the AOX1 promoter. For example, changes in the DO concentrations (DO spikes) allow you to determine whether all the glycerol is consumed from the culture before adding methanol. Secondly, it ensures that your methanol feed does not exceed the rate of consumption. Excess methanol (> 1-2% v/v) may be toxic.Manipulation of DO If carbon is limiting, shutting off the carbon source should cause the culture to decrease its metabolic rate, and the DO to rise (spike). Terminate the carbon feed and time how long it takes for the DO to rise 10%, after which the carbon feed is turned back on. If the lag time is short (< 1 minute), the carbon source is limiting.Fermenter Preparation and Glycerol Batch PhaseInoculum Seed Flask Preparation Remember not to put too much medium in the baffled flasks. Volume should be 10-30% of the total flask volume.1. Baffled flasks containing a total of 5-10% of the initial fermentation volume ofMGY or BMGY are inoculated with a colony from a MD or MGY plate or from a frozen glycerol stock.2. Flasks are grown at 30°C, 250-300 rpm, 16-24 hours until OD600= 2-6. Toaccurately measure OD600> 1.0, dilute a sample of your culture 10-fold before reading.Glycerol Batch Phase 1. Sterilize the fermenter with the Fermentation Basal Salts medium containing 4%glycerol (see page 11).2. After sterilization and cooling, set temperature to 30°C, agitation and aeration tooperating conditions (usually maximum rpm and 0.1-1.0 vvm air), and adjust the pH of the Fermentation Basal Salts medium to 5.0 with 28% ammonium hydroxide(undiluted ammonium hydroxide). Add aseptically 4.35 ml PTM1trace salts/liter of Fermentation Basal Salts medium.3. Inoculate fermenter with approximately 5-10% initial fermentation volume from theculture generated in the inoculum shake flasks. Note that the DO will be close to 100% before the culture starts to grow. As the culture grows, it will consumeoxygen, causing the DO to decrease. Be sure to keep the DO above 20% by adding oxygen as needed.4. Grow the batch culture until the glycerol is completely consumed (18 to 24 hours).This is indicated by an increase in the DO to 100%. Note that the length of timeneeded to consume all the glycerol will vary with the density of the initial inoculum.5. Sampling is performed at the end of each fermentation stage and at least twice daily.We take 10 ml samples for each time point, then take 1 ml aliquots from this 10 mlsample. Samples are analyzed for cell growth (OD600and wet cell weight), pH, microscopic purity, and protein concentrations or activity. Freeze the cell pellets and supernatants at -80°C for later analysis. Proceed to Glycerol Fed-Batch Phase,page 5.Yield A cellular yield of 90 to 150 g/liter wet cells is expected for this stage. Recombinant protein will not yet be produced due to the absence of methanol.Introduction Once all the glycerol is consumed from the batch growth phase, a glycerol feed isinitiated to increase the cell biomass under limiting conditions. When you are ready toinduce with methanol, you can use DO spikes to make sure the glycerol is limited.Glycerol Fed-Batch Phase 1. Initiate a 50% w/v glycerol feed containing 12 ml PTM1trace salts per liter of glycerol feed. Set the feed rate to 18.15 ml/hr /liter initial fermentation volume.2. Glycerol feeding is carried out for about four hours or longer (see below). A cellularyield of 180 to 220 g/liter wet cells should be achieved at the end of this stage while no appreciable recombinant protein is produced.Note The level of expressed protein depends on the cell mass generated during the glycerolfed-batch phase. The length of this feed can be varied to optimize protein yield. A rangeof 50 to 300 g/liter wet cells is recommended for study. A maximum level of 4%glycerol is recommended in the batch phase due to toxicity problems with higher levelsof glycerol.Important If dissolved oxygen falls below 20%, the glycerol or methanol feed should bestopped and nothing should be done to increase oxygen rates until the dissolvedoxygen spikes. At this point, adjustments can be made to agitation, aeration, pressure oroxygen feeding.Proteases In the literature, it has been reported that if the pH of the fermentation medium islowered to 3.0, neutral proteases are inhibited. If you think neutral proteases aredecreasing your protein yield, change the pH control set point to 3.0 during the glycerolfed-batch phase (above) or at the beginning of the methanol induction (next page) andallow the metabolic activity of the culture to slowly lower the pH to 3.0 over 4 to 5 hours(Brierley, et al., 1994; Siegel, et al., 1990).Alternatively, if your protein is sensitive to low pH, it has been reported that inclusion ofcasamino acids also decreases protease activity (Clare, et al., 1991).Introduction All of the glycerol needs to be consumed before starting the methanol feed to fullyinduce the AOX1 promoter on methanol. However, it has been reported that a "mixedfeed" of glycerol and methanol has been successful to express recombinant proteins(Brierley, et al., 1990; Sreekrishna, et al., 1989). It is important to introduce methanolslowly to adapt the culture to growth on methanol. If methanol is added too fast, it willkill the cells. Once the culture is adapted to methanol, it is very important to use DOspikes to analyze the state of the culture and to take time points over the course ofmethanol induction to optimize protein expression. Growth on methanol also generates alot of heat, so temperature control at this stage is very important.Mut+ Methanol Fed-Batch Phase 1. Terminate glycerol feed and initiate induction by starting a 100% methanol feedcontaining 12 ml PTM1trace salts per liter of methanol. Set the feed rate to 3.6 ml/hr per liter initial fermentation volume.2. During the first 2-3 hours, methanol will accumulate in the fermenter and thedissolved oxygen values will be erratic while the culture adapts to methanol.Eventually the DO reading will stabilize and remain constant.3. If the DO cannot be maintained above 20%, stop the methanol feed, wait for theDO to spike and continue on with the current methanol feed rate. Increaseagitation, aeration, pressure or oxygen feeding to maintain the DO above 20%. 4. When the culture is fully adapted to methanol utilization (2-4 hours), and is limitedon methanol, it will have a steady DO reading and a fast DO spike time (generally under 1 minute). Maintain the lower methanol feed rate under limited conditions for at least 1 hour after adaptation before doubling the feed. The feed rate is then doubled to ~7.3 ml/hr/liter initial fermentation volume.5 After 2 hours at the 7.3 ml/hr/liter feed rate, increase the methanol feed rate to~10.9 ml/hr per liter initial fermentation volume. This feed rate is maintainedthroughout the remainder of the fermentation.6. The entire methanol fed-batch phase lasts approximately 70 hours with a total ofapproximately 740 ml methanol fed per liter of initial volume. However, this may vary for different proteins.Note: The supernatant may appear greenish. This is normal.Yield The cell density can increase during the methanol fed-batch phase to a final level of 350 to 450 g/liter wet cells. Remember that because most of the fermentation is carried out ina fed-batch mode, the final fermentation volume will be approximately double the initialfermentation volume.Fermentation of Mut S Pichia Strains Since Mut S cultures metabolize methanol poorly, their oxygen consumption is very low. Therefore, you cannot use DO spikes to evaluate the culture. In standard fermentations of a Mut S strain, the methanol feed rate is adjusted to maintain an excess of methanol in the medium which does not exceed 0.3% (may be determined by gas chromatography). While analysis by gas chromatography will insure that nontoxic levels of methanol are maintained, we have used the empirical guidelines below to express protein in Mut S strains. A gas chromatograph is useful for analyzing and optimizing growth of Mut S recombinants.continued on next pageMethanol Fed-Batch Phase, continuedMut S Methanol Fed- Batch Phase The first two phases of the glycerol batch and fed-batch fermentations of the Mut S strains are conducted as described for the Mut+ strain fermentations. The methanol induction phases of the Mut+ and Mut S differ in terms of the manner and amount in which the methanol feed is added to the cultures.1. The methanol feed containing 12 ml PTM1trace salts per liter of methanol is initiated at 1 ml/hr/liter initial fermentation volume for the first two hours. It is then increased in 10% increments every 30 minutes to a rate of 3 ml/hr which ismaintained for the duration of the fermentation.2.. The vessel is then harvested after ~100 hours on methanol. This time may be variedto optimize protein expression.Harvesting and Lysis of CellsIntroduction The methods and equipment listed below are by no means complete. The amount of cells or the volume of supernatant will determine what sort of equipment you need.Harvesting Cells and Supernatant For small fermentations (1-10 liters), the culture can be collected into centrifuge bottles (500-1000 ml) and centrifuged to separate the cells from the supernatant.For large fermentations, large membrane filtration units (Millipore) or a Sharples centrifuge can be used to separate cells from the supernatant. The optimal method will depend on whether you need the cells or the supernatant as the source of your protein and what you have available.Supernatants can be loaded directly onto certain purification columns or concentrated using ultrafiltration.Cell Lysis We recommend cell disruption using glass beads as described in Current Protocols inMolecular Biology, page 13.13.4. (Ausubel, et al., 1990) or Guide to ProteinPurification (Deutscher, 1990). This method may be tedious for large amounts of cells.For larger amounts, we have found that a microfluidizer works very well. Frenchpressing the cells does not seem to work as well as the glass beads or the microfluidizer.ReferencesIntroduction Most of the references below refer to papers where fermentation of Pichia wasperformed. Note that some of these are patent papers. You can obtain copies of patentsusing any of the following methods.• Patent Depository Libraries. U. S. patents and international patents granted underthe Patent Cooperation Treaty (PCT) are available on microfilm. These can be copiedand mailed or faxed depending on length. There is a fee for this service. The referencelibrarian at your local library can tell you the location of the nearest Patent DepositoryLibrary.• Interlibrary Loan. If you are not near a Patent Depository Library, you may request acopy of the patent through interlibrary loan. There will be a fee for this service.• U. S. Patent Office. Requests may be made directly to the Patent Office, Arlington,VA. Please call 703-557-4636 for more information on cost and delivery.• Private Library Services. There are private companies who will retrieve and sendyou patents for a fee. Two are listed below:Library Connection: 804-758-3311Rapid Patent Services: 800-336-5010Citations Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,Struhl, K., eds (1990) Current Protocols in Molecular Biology. GreenePublishing Associates and Wiley-Interscience, New York.Brierley, R. A., Siegel, R. S., Bussineau, C. M. Craig, W. S., Holtz, G. C., Davis, G. R.,Buckholz, R. G., Thill, G. P., Wondrack, L. M., Digan, M. E., Harpold, M. M.,Lair, S. V., Ellis, S. B., and William, M. E. (1989) Mixed Feed RecombinantYeast Fermentation. International Patent (PCT) Application. Publication No.WO 90/03431.Brierley, R. A., Bussineau, C., Kosson, R., Melton, A., and Siegel, R. S. (1990)Fermentation Development of Recombinant Pichia pastoris Expressing theHeterologous Gene: Bovine Lysozyme. Ann. New York Acad. Sci.589: 350-362.Brierley, R. A., Davis, G. R. and Holtz, G. C. (1994) Production of Insulin-Like GrowthFactor-1 in Methylotrophic Yeast Cells. United States Patent5,324,639.Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M.M., Sreekrishna, K. and Henwood, C. A. (1991) Production of EpidermalGrowth Factor in Yeast: High-level Secretion Using Pichia pastoris StrainsContaining Multiple Gene Copies. Gene105: 205-212.Cregg, J. M., Tschopp, J. F., Stillman, C., Siegel, R., Akong, M., Craig, W. S.,Buckholz, R. G., Madden, K. R., Kellaris, P. A., Davis, G. R., Smiley, B. L.,Cruze, J., Torregrossa, R., Veliçelebi, G. and Thill, G. P. (1987) High-levelExpression and Efficient Assembly of Hepatitis B Surface Antigen in theMethylotrophic Yeast Pichia pastoris. Bio/Technology5: 479-485.Cregg, J. M., Vedvick, T. S. and Raschke, W. C. (1993) Recent Advances in theExpression of Foreign Genes in Pichia pastoris. Bio/Technology11: 905-910.Deutscher, M. P. (1990) Guide to Protein Purification. In: Methods in Enzymology (J.N. Abelson and M. I. Simon, eds.) Academic Press, San Diego, CA.continued on next pageReferences, continuedCitations, continuedDigan, M. E., Lair, S. V., Brierley, R. A., Siegel, R. S., Williams, M. E., Ellis, S. B., Kellaris, P. A., Provow, S. A., Craig, W. S., Veliçelebi, G., Harpold, M. M. andThill, G. P. (1989) Continuous Production of a Novel Lysozyme via Secretionfrom the Yeast Pichia pastoris. Bio/Technology7: 160-164.Hagenson, M. J., Holden, K. A., Parker, K. A., Wood, P. J., Cruze, J. A., Fuke, M., Hopkins, T. R. and Stroman, D. W. (1989) Expression of Streptokinase inPichia pastoris Yeast. Enzyme Microbiol. Technol.11: 650-656.Laroche, Y., Storme, V., Meutter, J. D., Messens, J. and Lauwereys, M. (1994) High-Level Secretion and Very Efficient Isotopic Labeling of Tick AnticoagulantPeptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris.Bio/Technology12: 1119-1124.Romanos, M. A., Clare, J. J., Beesley, K. M., Rayment, F. B., Ballantine, S. P., Makoff,A. J., Dougan, G., Fairweather, N. F. and Charles, I. G. (1991) RecombinantBordetella pertussis Pertactin p69 from the Yeast Pichia pastoris High LevelProduction and Immunological Properties. Vaccine9: 901-906.Siegel, R. S. and Brierley, R. A. (1989) Methylotrophic Yeast Pichia pastoris Produced in High-cell-density Fermentations With High Cell Yields as Vehicle forRecombinant Protein Production. Biotechnol. Bioeng.34: 403-404.Siegel, R. S., Buckholz, R. G., Thill, G. P., and Wondrack, L. M. (1990) Production of Epidermal Growth Factor in Methylotrophic Yeast Cells. International Patent(PCT) Application. Publication No. WO 90/10697.Sreekrishna, K., Nelles, L., Potenz, R., Cruse, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., Wood, P. and Parker, K. (1989) High LevelExpression, Purification, and Characterization of Recombinant Human TumorNecrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris.Biochemistry28(9): 4117-4125.©2002 Invitrogen Corporation. All rights reservedRecipesFermentation Basal Salts Medium For 1 liter, mix together the following ingredients:Phosphoric acid, 85% (26.7 ml)Calcium sulfate 0.93 gPotassium sulfate 18.2 gMagnesium sulfate-7H2O 14.9gPotassium hydroxide 4.13 gGlycerol 40.0g Water to 1 literAdd to fermenter with water to the appropriate volume and sterilize.PTM1 Trace Salts Mix together the following ingredients:Cupric sulfate-5H2O 6.0gSodium iodide 0.08 gManganese sulfate-H2O 3.0gSodium molybdate-2H2O 0.2gBoric Acid 0.02 g Cobalt chloride 0.5 g Zinc chloride 20.0 gFerrous sulfate-7H2O 65.0gBiotin 0.2gSulfuric Acid 5.0 mlWater to a final volume of 1 literFilter sterilize and store at room temperature.Note: There may be a cloudy precipitate upon mixing of these ingredients. Filter-sterilize as above and use.11。

毕赤酵母表达蛋白步骤

毕赤酵母表达蛋白步骤一、引言毕赤酵母（Pichia pastoris）是一种常用的真菌表达系统，被广泛应用于蛋白质的表达和生物技术研究中。

其优势包括高表达水平、易于培养和操作、能够正确折叠复杂蛋白等。

本文将介绍毕赤酵母表达蛋白的步骤。

二、构建表达载体毕赤酵母表达系统的关键是表达载体的构建。

首先，需要选择适合的表达载体，常用的有pPIC6、pPICZα等。

然后，在载体上选择合适的启动子和信号序列，以确保蛋白质能够被正确表达和分泌。

同时，还需要在表达载体上加入选择标记，如His标签、FLAG标签等，以便后续的蛋白质纯化和检测。

三、转化毕赤酵母将构建好的表达载体转化入毕赤酵母中，使其成为表达宿主。

转化方法包括电击转化、化学转化等。

其中，电击转化是常用的方法，通过电击脉冲使毕赤酵母细胞膜发生破裂，使表达载体进入细胞内。

转化后，将细胞培养在选择性培养基上，筛选出带有表达载体的毕赤酵母克隆。

四、表达蛋白经过转化筛选后，得到含有目标蛋白表达载体的毕赤酵母克隆。

接下来，需要将克隆进行培养，在适当的条件下诱导蛋白的表达。

常用的诱导剂包括甲醇、巯基乙醇等，通过加入适量的诱导剂，可以使目标蛋白得到高效表达。

五、蛋白纯化在蛋白表达后，需要进行蛋白纯化，以获得纯度较高的目标蛋白。

常用的纯化方法包括亲和层析、离子交换层析、凝胶过滤层析等。

在选择纯化方法时，需要根据目标蛋白的性质和需求进行合理选择。

同时，可以利用加入的选择标记，如His标签，通过亲和层析纯化进行快速高效的纯化。

六、蛋白鉴定和功能分析蛋白纯化后，需要进行蛋白的鉴定和功能分析。

常用的鉴定方法包括SDS-PAGE、Western blot等，可以确定蛋白的分子量和纯度。

功能分析则可以通过生物学实验来进行，如酶活测定、结合实验等，以验证目标蛋白的功能。

七、应用和展望毕赤酵母表达系统在生物技术和蛋白质研究领域有着广泛的应用。

通过该系统，可以高效表达各种蛋白，包括抗体、酶和重组蛋白等。

GS115毕赤酵母表达菌使用说明

GS115 毕赤酵母表达菌
编号
名称
北京华越洋生物 NRR01030 GS115 毕赤酵母表达菌
基本信息：
名称：GS115 毕赤酵母表达菌
规格：300ul 甘油菌
储存温度： -‐80℃
发突变为组氨酸野生型的概率一般低于 10-‐8。GS115 毕赤酵母可以在 YPD
培养基中生长，或者在补充有组氨酸的 minimal media 中生长，但是无法
在单独的 minimal media 中生长。GS115 毕赤酵母在做质粒转化的时候，
可采用电转化的方式将质粒转入。
基因组：
His4（基因 5 是毕赤酵母菌株，是巴斯德毕赤酵母的一种，属于真核细胞。
一般的针对原核生物的抗生素例如卡那和氨苄对酵母是无效的，因此为了
操作说明：
1，本品包含一份甘油菌，使用本甘油菌时可以不用完全融解，在甘油菌表
面蘸取少量涂板或进行液体培养即可。也可以完全融解后使用，但随着冻融次数
的增加，细菌的活力会逐渐下降。
2，为保证菌种纯正，避免其它细菌污染，尽量先划平板，然后再挑单克隆
菌落进行后续操作。
毕赤酵母适宜的生长温度是 28 至 30 度，温度超过 32 度对蛋白的表
达是有害的，并可能导致细胞的死亡。GS115 毕赤酵母是是组氨酸缺陷型
（His4 基因型），如果表达载体上携带有组氨酸基因，可补偿宿主菌的组
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
养。细菌在 30-‐35℃培养箱中培养 24-‐48h，真菌在 23-‐28℃培养箱中培养 24-‐72h
（必要时，可适当延长培养时间）。

毕赤酵母表达操作手册(PDF精译版)

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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。

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。

毕赤酵母的摇瓶发酵方法[指南]

毕赤酵母的摇瓶发酵方法：一、摇瓶发酵方法:毕赤酵母摇瓶发酵方法分为两个阶段，1、酵母菌株生长阶段；2、脂肪酶诱导表达阶段。

1、酵母生长阶段。

准备试剂：1000ml BMGY培养基，1000ml BMMY培养基，10X的甲醇，摇瓶1L（灭菌），温控摇床，50ml离心管（灭菌）。

紫外分光光度计，石英比色皿。

以下所有操作均在超净台内或者无菌条件下完成。

（1）往灭好菌的IL摇瓶中加入100mlBMGY培养基，然后加入约1ml脂肪酶菌株（培养基：菌液=100：1），用透气膜封口（透气，但是细菌不能透过）。

置于温控摇床上，温度调至300C，转速为250-300rpm/min，使酵母生长，OD600=2.0-6.0，时间约为15-24小时。

（2）将发酵液转入50ml离心管，1500g-3000g离心5min。

去掉上清，用BMMY 培养基将菌体浓度稀释至OD600=1.0，约有500ml左右。

将稀释后的发酵液分别加入到1L的药瓶中，每个摇瓶150ml发酵液（绝不能超过200ml）。

（3）将摇瓶置于温控摇床上，温度调至300C，转速为250-300rpm/min，使酵母表达脂肪酶，每24小时加入一次5%的甲醇，使甲醇的终浓度为0.5%。

连续诱导表达48小时。

（4）将发酵液进行12000rpm/min离心5min，取上清（若上清仍混浊，可反复离心）；进行酶活分析和蛋白含量分析。

BMGY培养基的配制（1000ml）：20g蛋白胨（peptone）,10g酵母提取物（Yeast Extract）,加水至700ml；1210C高温灭菌20min。

然后分别在无菌条件下加入10X YNB 100ml，10X 磷酸钾缓冲液（PH6.0）100ml，10X甘油 100ml。

BMMY培养基的配制方法（1000ml）：20g蛋白胨（peptone）,10g酵母提取物,加水至700ml；1210C高温灭菌20min。

然后分别在无菌条件下加入10X YNB 100ml，10X 磷酸钾缓冲液（PH6.0）100ml，10X甲醇 100ml。

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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。

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。

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。

而调控产生醇过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。

两种醇氧化酶蛋白：毕赤酵母中有两个基因编码醇氧化酶－AOX1及AOX2。

细胞中大多数的醇氧化酶是AOX1基因产物。

甲醇可紧密调节、诱导AOX1基因的高水平表达，较典型的是占可溶性蛋白的30%以上。

AOX1基因已被分离，含AOX1启动子的质粒可用来促进编码外源蛋白的目的基因的表达。

AOX2基因与AOX1基因有97%的同源性，但在甲醇中带AOX2基因的菌株比带AOX1基因菌株慢得多，通过这种甲醇利用缓慢表型可分离Muts菌株。

表达：AOX1基因的表达在转录水平受调控。

在甲醇中生长的细胞大约有5%的polyA+ RNA 来自AOX1基因。

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，仍不足以使AOX1基因达到最低水平的表达，诱导物甲醇是AOX1基因可辨表达水平所必需的。

AOX1突变表型：缺失AOX1基因，会丧失大部分的醇氧化酶活性，产生一种表型为Muts的突变株（methanol utilization slow），过去称为Mut，而Muts可更精确地描述突变子的表型。

结果细胞代谢甲醇的能力下降，因而在甲醇培养基中生长缓慢。

Mut+（methanol utilization plus）指利用甲醇为唯一碳源的野生型菌株。

这两种表型用来检测外源基因在毕赤酵母转化子中的整合方式。

蛋白胞内及分泌表达：外源蛋白可在毕赤酵母胞内表达或分泌至胞外。

分泌表达需要蛋白上的信号肽序列，将外源蛋白靶向分泌通路。

几种不同的分泌信号序列已被成功应用，包括几种外源蛋白本身分泌信号序列，利用酿酒酵母α因子前原肽信号序列也获得许多成功。

分泌表达外源蛋白的最大优点是：毕赤酵母只分泌很少的自身蛋白，加上毕赤酵母最小生长培养基中只有少量的蛋白，这意味着分泌的外源蛋白是培养基中蛋白的主要组成成份，也可算作蛋白纯化的第一步。

注意，如果外源蛋白一级结构中有可识别的糖基化位点（Asn-X-Ser/Thr）,则这些位点可能发生糖基化。

翻译后修饰：与酿酒酵母相比，毕赤酵母在分泌蛋白的糖基化方面有优势，因为不会使其过糖基化。

酿酒酵母与毕赤酵母大多数为N-连接糖基化高甘露糖型，然而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。

另外，酿酒酵母核心寡糖有末端α-1,3聚糖连接头，而毕赤酵母则没有。

一般认为酿酒酵母中糖基化蛋白的α-1,3聚糖接头与蛋白的超抗原性有关，使得这些蛋白不适于治疗应用。

虽然未经证明，但这对毕赤酵母产生的糖蛋白不构成问题，因为毕赤酵母表达蛋白与高级真核生物糖蛋白结构相似。

选择载体用于基因多拷贝整合：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蛋白的表达量。

该试剂盒中的三个载体均可用于在体内（pPIC3.5K, pPIC9K）或体外（pAO815）产生并分离多拷贝插入，同时可检测增加重组基因的拷贝数是否增加蛋白表达量。

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

pPIC3.5K, pAO815用于胞内表达，而pPIC9K用于分泌表达，所有载体均利用AOX1启动子来诱导高水平表达。

多拷贝插入频率：毕赤酵母His+转化子高拷贝整合事件自发发生的概率为1－10%，体内方法可筛选可能插入多拷贝外源基因的His+转化子，体外方法可通过连接构建多拷贝子。

当选择His+转化子时，它们中插入体外构建结构多聚体的概率很高。

体内多拷贝插入的产生：Ppic3.5k及Ppic9k含有细菌kan基因，赋予毕赤酵母遗传霉素抗性，注意Kan并不赋予毕赤酵母卡那霉素抗性。

遗传霉素抗性水平主要依赖整合的kan基因的数目。

单拷贝Ppic3.5k或Ppic9k整合入毕赤酵母基因组后，赋予毕赤酵母约0.25mg/ml的遗传霉素抗性水平。

任何载体多拷贝整合可增加遗传霉素抗性水平，从0.5mg/ml（1-2拷贝）到4mg/ml（7-12拷贝）。

由于kan基因与表达盒（pAOX1及目的基因）之间有遗传连锁，可从遗传霉素高抗性推断该克隆所包含多拷贝目的基因数。

由于基因的剂量效益，蛋白的表达可能会增加。

因此，kan基因可检测转化子是否含有多拷贝目的基因。

下图显示多拷贝插入及kan基因与表达盒的连锁。

遗传霉素直接选择：在酵母中对遗传霉素抗性进行直接选择并不十分有效，因为新转化的细胞需要时间表达足够量的抗性因子。

由于酵母生长比细菌慢得多，大部分重组酵母在积累足够多的抗性因子以抵抗平板上抗生素之前就已经被杀死了。

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

虽然可以用电泳进行直接筛选，但用在遗传霉素筛选之后再进行电泳筛选，获得含高拷贝克隆的机会更大，大约可获得5-9拷贝的克隆，而直接电泳选择只能获得平均为1-3拷贝的克隆。

原生质转化时不能用遗传霉素直接选择。

体外多拷贝插入的产生：下图显示如何产生多表达盒插入载体以转化毕赤酵母。

目的基因插入独个EcoRI位点后，产生的表达盒（pAOX1及目的基因）上下游侧翼分别为独个的BglII及BamHI位点。

含目的基因的pAOX815用BglII及BamHI消化以分离表达盒，表达盒再插入BamHI 位点以产生串联重复表达盒，重复该插入程序可产生一系列含单个HIS4基因及逐渐增加数目表达盒的载体。

用体外形成的多拷贝子转化毕赤酵母增加了多拷贝表达盒重组子出现的频率，可设计包含一特定数目多拷贝插入的毕赤酵母重组子。

转化及整合：可产生两个不同表型的His+重组菌株：质粒DNA线性化位置不同，转化GS115后可产生两种转化子His+Mut+及His+Muts。

KM71只产生His+Muts，因为该菌株为Muts表型。

两种重组子Mut＋及Muts都是有用的，因为一个表型可能比另一个表型更有利于蛋白表达。

理想条件下，每一个表型应该检测6-10个重组子。

没有办法预测哪个结构或克隆更利于蛋白表达。

强烈推荐用PCR分析重组子来证实整合情况。

成功将基因构建至AOX1启动子下游后，线性化质粒转化毕赤酵母时激发重组。

下图显示用不同酶消化时产生何种重组子。

限制酶插入事件GS115表型KM71表型SalI或StuI 插入his4 His+Mut+ His+MutsSacI 插入5’AOX1 His+Mut+ His+MutsBglII 取代AOX1 His+Muts His+Muts（不推荐）表达及扩大培养：用PCR证实毕赤酵母重组后，可检测His+Mut+及His+Muts的表达。

小规模培养每个重组子，用甲醇诱导，检测时间点.如果是胞内表达，每个时间点细胞沉淀用SDS-PAGE分析;如果是分泌表达，分析每个时间点的细胞及上清。

如果有蛋白的抗体，推荐既用考马斯亮蓝染色又用western blot分析SDS-PAGE凝胶。

如果可以,建议检测蛋白活性。

因为并不是所有蛋白都能达到g/l的水平，所以建议进行western blot或活性分析，不要仅做SDS-PAGE 考马斯亮蓝染色分析。

如何选择最佳的表达蛋白毕赤酵母菌株及优化诱导见P49-50。

如表达已达最优，大规模表达以产生更多蛋白。

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

GS115及KM71自发回复突变到His＋原养生物机率小于1/108。

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基因破坏AOX1基因后，产生KM71 MutsArg+His-菌株。

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

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

KM71结构：ARG4基因（约2kb）插入到克隆的野生型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。

ARG4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Arg+转化子并分析Muts表型。

Arg+转化子遗传分析显示野生型AOX1被aox1::ARG4结构所取代。

重点：用KM71的优点是，不需要在甲醇最小培养基中筛选Mut表型。

所有转化子都是Muts 表型。

第二，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。

不幸的是，仅添加精氨酸并不能完全缓和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。

菌株表达对照：GS115/His+Muts白蛋白：该菌株为筛选毕赤酵母分泌表达转化子与Muts表型时的对照。

血清白蛋白基因及其自身分泌信号被整合进毕赤酵母AOX1位点。