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查看更多产品信息 Pichia methanolica Expression System - FAQs (K178001)
14 个常见问题解答
Pichia methanolica 表达系统已停产。
我们建议将酵母置于15%甘油中保存在–80°C。甘油储液可长期保存（除非经过多次冻融）。在制备甘油储液时，我们建议使用过夜培养物并将其浓缩2-4倍。将细胞离心，并使用原始体积25–50%的甘油/培养基重悬。最好使用新鲜培养基加甘油冻存细胞，而不仅仅是将甘油加到过夜培养物中这么简单。
我们提供original Pichia pastoris表达系统、PichiaPink表达系统和Saccharomyces cerevisiae酿酒酵母表达系统，用于重组蛋白表达。已知P. pastoris和S. cerevisiae遗传性质已十分明确，均可进行多种翻译后修饰。
P. pastoris毕赤酵母表达系统结合了大肠杆菌表达（高水平表达、易于扩大规模和低成本）和真核系统表达（蛋白加工、折叠和翻译后修饰）的优势，从而可对有功能活性的重组蛋白进行高水平生产。作为一种酵母，毕赤酵母Pichia pastoris与酿酒酵母Saccharomyces cerevisiae具有相似的分子和基因操作优势，而且其外源蛋白表达水平比酿酒酵母Saccharomyces cerevisiae高10-100倍。这些特性使毕赤酵母Pichia pastoris 非常适合用作蛋白表达系统。Pichia表达载体含有强乙醇氧化酶（AOX1）启动子，用于高水平、严格调控的诱导型的表达；或者含有甘油醛-3-磷酸脱氢酶（GAP）启动子，用于高水平的组成型表达。诱导型和组成型表达构建体均整合到P. pastoris基因组，建立蛋白表达水平极高的稳定宿主，特别是在使用发酵器的情况下。我们可提供的Pichia pastoris表达系统包括：
•PichiaPink酵母表达系统：最新的Pichia pastoris表达系统包含低拷贝和高拷贝质粒骨架、8种分泌信号序列和4种酵母菌株，这些有助于优化得到最高的重组蛋白产率。所有PichiaPink载体都含有AOX1启动子，用于高水平诱导型表达；还含有ADE2标记物，利用ADE2互补作用筛选转化株（即腺嘌呤缺陷型的互补）而不是抗生素筛选。但是，它们通过不同长度的启动子表达ADE2基因产物，从而决定了整合质粒的拷贝数。在pPink-LC载体中，ADE2标记物的启动子为82 bp，可提供低拷贝表达；在pPink-HC载体，ADE2标记物的启动子为13 bp，可提供高拷贝表达。该系统也可诱导pPinkα-HC载体（含S. cerevisiae α-交配因子前导序列）产生高拷贝数分泌表达，并且还包含8种分泌信号序列可优化分泌表达。
•EasySelect Pichia表达试剂盒：一种传统Pichia 表达试剂盒，包含pPICZ和pPICZα载体，可分别用于目的基因的细胞内和分泌表达。这些载体含有AOX1启动子，可产生高水平的诱导型表达；还含有Zeocin抗生素抗性标记物，可直接进行多拷贝整合载体的筛选。它们有助于对表达的蛋白进行简单的亚克隆、纯化以及快速检测。
•Original Pichia表达试剂盒：该试剂盒包含pPIC9、pPIC3.5、pHIL-D2和pHIL-S1载体，每种载体都含有AOX1启动子，可产生高水平的诱导型表达；还含有HIS4基因，用于在缺乏组氨酸的培养基上筛选his4酵母株。pPIC9带有S. cerevisiae α-因子分泌信号，而pHIL-S1带有Pichia pastoris碱性磷酸酶信号序列（PHO），可指导蛋白质向培养基的转运。pHIL-D2和pPIC3.5专为细胞内表达而设计。
•多拷贝Pichia表达试剂盒：该试剂盒专为最大化表达而设计，包含pPIC3.5K、pPIC9K和pAO815载体，可产生和选择含多个目的基因的Pichia菌株。它们可通过体内方法（pPIC3.5K和pPIC9K）或体外方法（pAO815）分离和生成多拷贝插入片段。所有这些载体都含有AOX1启动子，可产生高水平的诱导型表达；还含有HIS4基因，用于在缺乏组氨酸的培养基上筛选his4菌株。pPIC9K载体可指导表达蛋白的分泌，而从pPIC3.5K和pAO815载体表达的蛋白仍留在细胞内。pPIC9K和pPIC3.5K载体带有卡那霉素抗性标记物，从而使Pichia对Geneticin试剂产生抗性。可通过Geneticin试剂抗性水平高低来鉴定自发的多次插入事件。在缺乏组氨酸的培养基上对Pichia转化株进行筛选，并筛选它们对Geneticin试剂的抗性水平。在高浓度Geneticin试剂中的生长能力，表示多拷贝的卡那霉素抗性基因以及目的基因被整合到了基因组。
为实现在S. cerevisiae中的表达，我们提供pYES 载体系列。每个pYES载体都带有GAL1基因的启动子和增强子序列，可实现诱导型表达。GAL1启动子是使用最广泛的酵母启动子之一，因为它在半乳糖的诱导下具有很强的转录活性。pYES载体还具有2 µ复制起点，可以维持游离的高拷贝数（10-40拷贝/细胞）。
酵母是一种单细胞的真核生物，在成分确定培养基中可快速生长（在含葡萄糖培养基中的倍增时间通常为2.5小时），相比使用昆虫或哺乳细胞生产重组蛋白更简单、更便宜（见下表）。这些良好的特性使酵母适用于从多孔培养板、摇瓶和持续搅拌槽生物反应器到小型试验工厂和工业规模反应器等多种形式的蛋白制备。
实验室最常用的酵母种类是酿酒酵母（Saccharomyces cerevisiae，又名Baker或Brewer酵母）和一些毕赤酵母属（Pichia）的甲醇营养型酵母。S. cerevisiae和P. pastoris的遗传性质均已明确，并能够对蛋白进行翻译后修饰，包括二硫键形成和糖基化，这对于一些重组蛋白发挥正常功能具有重要作用。但是，应注意酵母的糖基化与哺乳细胞的有所不同：在S. cerevisiae中，O-连接的寡糖只有甘露糖残基，而更高等的真核蛋白有唾液酸化的O-连接糖链。此外，已知S. cerevisiae可过度糖基化N-端位点，从而导致蛋白质结合和活性发生改变，并可能在治疗应用中产生异常的免疫原应答。在P. pastoris中，寡糖链的长度短很多，并且已有一个P. pastoris株被报导可产生复杂的、末端唾液酸化的或“人源化”的糖蛋白。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
The Pichia methanolica Expression System has been discontinued. As an alternative, we offer the Pichia Pink Expression System (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/yeast-protein-expression/pichiapink-yeast-expression-systems.html)
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We recommend storing yeast frozen at -80 degrees C in 15% glycerol. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws). When making a glycerol stock, we recommend using an overnight culture and concentrating it 2-4 fold. Spin down cells and suspend in 25-50% of the original volume with glycerol/medium. It is better to store frozen cells in fresh medium plus glycerol, rather than simply adding glycerol into the overnight culture.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We offer the original Pichia pastoris expression systems, PichiaPink expression system, and Saccharomyces cerevisiae yeast expression system for expression of recombinant proteins. Both P. pastoris and S. cerevisiae have been genetically well-characterized and are known to perform many posttranslational modifications.
The P. pastoris expression system combines the benefits of expression in E. coli (high-level expression, easy scale-up, and inexpensive growth) and the advantages of expression in a eukaryotic system (protein processing, folding, and posttranslational modifications), thus allowing high-level production of functionally active recombinant protein. As a yeast, Pichia pastoris shares the advantages of molecular and genetic manipulations with Saccharomyces cerevisiae, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia pastoris very useful as a protein expression system. The Pichia expression vectors contain either the powerful alcohol oxidase (AOX1) promoter for high-level, tightly controlled expression, or the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for high-level, constitutive expression. Both inducible and constitutive expression constructs integrate into the P. pastoris genome, creating a stable host that generates extremely high protein expression levels, particularly when used in a fermentor. The Pichia pastoris expression systems we offer include:
- PichiaPink Yeast Expression System: Newer Pichia pastoris expression system that contains both low- and high-copy plasmid backbones, 8 secretion signal sequences, and 4 yeast strains to help optimize for the highest yield possible of the recombinant protein. All PichiaPink vectors contain the AOX1 promoter for high-level, inducible expression and the ADE2 marker for selecting transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. However, they express the ADE2 gene product from promoters of different lengths, which dictate the copy number of the integrated plasmids. The pPink-LC vector has an 82 bp promoter for the ADE2marker and offers low-copy expression, and the pPink-HC vector has a 13 bp promoter for the ADE2marker and offers high-copy expression. The system also includes the pPinkalpha-HC vector (containing S. cerevisiae alpha-mating factor pre-sequence) for high copy number secreted expression, and provides eight secretion signal sequences for optimization of secreted expression.
- EasySelect Pichia Expression Kit: One of the original Pichia expression kits that contains the pPICZ and pPICZalpha vectors, for intracellular and secreted expression, respectively, of the gene of interest. These vectors contain the AOX1 promoter for high-level, inducible expression and the Zeocin antibiotic resistance marker for direct selection of multi-copy integrants. They facilitate simple subcloning, simple purification, and rapid detection of expressed proteins.
- Original Pichia Expression Kit: The kit includes the pPIC9, pPIC3.5, pHIL-D2, and pHIL-S1 vectors, each of which carries the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. pPIC9 carries the S. cerevisiae alpha-factor secretion signal while pHIL-S1 carries the Pichia pastoris alkaline phosphatase signal sequence (PHO) to direct transport of the protein to the medium. pHIL-D2 and pPIC3.5 are designed for intracellular expression.
- Multi-Copy Pichia Expression Kit: This kit is designed to maximize expression and contains the pPIC3.5K, pPIC9K, and pAO815 vectors, which allow production and selection of Pichia strains that contain more than one copy of the gene of interest. They allow isolation and generation of multicopy inserts by in vivo methods (pPIC3.5K and pPIC9K) or in vitro methods (pAO815). All of these vectors contain the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K and pAO815 remain intracellular. The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance marker that confers resistance to Geneticin Reagent in Pichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin Reagent. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin Reagent. The ability to grow in high concentrations of Geneticin indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome.
- For expression in S. cerevisiae, we offer the pYES Vector Collection. Each pYES vector carries the promoter and enhancer sequences from the GAL1 gene for inducible expression. The GAL1 promoter is one of the most widely used yeast promoters because of its strong transcriptional activity upon induction with galactose. pYES vectors also carry the 2m origin and are episomally maintained in high copy numbers (10-40 copies per cell).
Yeast is a single-celled, eukaryotic organism that can grow quickly in defined media (doubling times are typically 2.5 hr in glucose-containing media) and is easier and less expensive to use for recombinant protein production than insect or mammalian cells (see table below). These positive attributes make yeast suitable for use in formats ranging from multi-well plates, shake flasks, and continuously stirred tank bioreactors to pilot plant and industrial-scale reactors.
The most commonly employed species in the laboratory are Saccharomyces cerevisiae (also known as Baker's or Brewer's yeast) and some methylotrophic yeasts of the Pichia genus. Both S. cerevisiae and P. pastoris have been genetically characterized and shown to perform the posttranslational disulphide bond formation and glycosylation that is crucial for the proper functioning of some recombinant proteins. However, it is important to note that yeast glycosylation does differ from that in mammalian cells: in S. cerevisiae, O-linked oligosaccharides contain only mannose moieties, whereas higher eukaryotic proteins have sialylated O-linked chains. Furthermore S. cerevisiae is known to hyperglycosylate N-linked sites, which can result in altered protein binding, activity, and potentially yield an altered immunogenic response in therapeutic applications. In P. pastoris, oligosaccharides are of much shorter chain length and a strain has been reported that can produce complex, terminally sialylated or “humanized” glycoproteins.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
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The use of antibiotics is not recommended because most antibiotics become inactivated at the low pH of the medium during Pichia fermentation. In other words, addition of antibiotics such as Ampicillin or Kanamycin won't hurt the fermentation process, but because of the low pH the antibiotics become inactivated or may even precipitate out. For best results, use good sterile techniques.
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Yes, Pichia should be done under BSL-1 conditions. BSL-1 is the lowest biosafety level.
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Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.