Name: expression of recombinant proteins in the methylotrophic yeast pichia pastoris
Uploaded: 2010-02-25T16:50:00
Description: Watch this Scientific Journal Video about Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris at JoVE.com

我们要感谢支持这项工作的创新，不列颠哥伦比亚省知识发展基金和卫生研究所（CIHR）加拿大研究所，加拿大基金会。 MT是从微生物的多样性和进化（CMDE）图拉基金会资助中心的奖学金支持。

重组蛋白在甲醇毕赤酵母中的表达在开始此协议，你应该有你感兴趣的基因在体育帧克隆酵母父载体和它测序检查的正确插入的基因载体的利益。 第一步：生成electrocompetent酵母细胞，线性的兴建和改造成体育酵母 对于这一步，你将需要有以下媒体和板： <ul><li> YPDS板</li><li> 600毫升YPD培养基</li><li>冰冷的无?...

<ol>
	<li>Cereghino, G. P., Cregg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+yeast+in+biotechnology:+protein+production+and+genetic+analysis.">Applications of yeast in biotechnology: protein production and genetic analysis.</a> Curr Opin Biotechnol. 10, 422-427 (1999).</li><li>Cregg, J. M., Barringer, K. J., Hessler, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pichia+pastoris+as+a+Host+System+for+Transformations.">Pichia pastoris as a Host System for Transformations.</a> Mol. Cell. Biol. 5, 3376-3385 (1985).</li><li>Cregg, J. M., Cereghino, J. L., Shi, J., Higgins, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+in+Pichia+pastoris.">Recombinant protein expression in Pichia pastoris.</a> Mol Biotechnol. 16, 23-52 (2000).</li><li>Cereghino, G. P., Cereghino, J. L., Ilgen, C., Cregg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+in+fermenter+cultures+of+the+yeast+Pichia+pastoris.">Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris.</a> Curr Opin Biotechnol. 4, 329-332 (2002).</li></ol>

蛋白的表达，使用甲醇毕赤酵母作为主机系统，是在本议定书。蛋白可分泌到根据使用的克隆载体介质。重组蛋白的分泌，使后续的纯化更容易。然而，所显示的条件可能要为不同的蛋白质和条件和表达时间的变化表达优化，可能导致蛋白质水平增加。在本议定书中所使用的向量有一个c - myc基因的抗原表位，并可以购买此抗原表位的抗体的功能。其中有没有抗体，这使得蛋白质的分...

expression of recombinant proteins in the methylotrophic yeast pichia pastoris

蛋白质在微生物的真核宿主毕赤酵母中的表达提供了可能产生一个快速且易于使用的表达系统的重组蛋白的高含量。 作为一种单细胞微生物体育酵母是容易操纵的，并在高细胞密度的廉价媒体迅速成长。作为真核细胞，P。酵母是能够执行许多更高的真核细胞中获得的重组蛋白进行翻译后修饰进行蛋白质折叠，蛋白加工，形成二硫键和糖基化[1 ]。 由于甲醇酵母P.酵母是甲醇作为唯一的碳源代谢。强启动醇氧化酶AOX1，严格监管和甲醇诱导和它感兴趣的基因的表达。因此，外源蛋白的表达可诱导培养基中[2加入甲醇; 3]。 另一个重要优势是重组蛋白分泌到培养基中，使用一个信号序列为目标的外源蛋白毕赤酵母的分泌途径。媒体酵母本身分泌的内源性蛋白和媒体没有添加蛋白质水平低，外源蛋白建立在介质中的总蛋白的多数，有利于蛋白质纯化步骤[3，4]。 这里所用的载体（pPICZαA）含有AOX1启动子的严格监管，甲醇诱导感兴趣的基因的表达，一个可供选择的Zeocin 抗性都大肠杆菌基因重组蛋白，分泌的α-因子的分泌信号大肠杆菌和毕赤酵母和C -末端肽含c - myc基因的抗原表位和多聚组氨酸（6xHis）检测和纯化重组蛋白的标记。我们也使用特定的抗 myc - HRP抗体承认父向量的C - myc基因的抗原表位的重组蛋白质免疫印迹分析。

Watch this Scientific Journal Video about Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris at JoVE.com

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

协议描述使用甲醇酵母蛋白的表达<em&gt;毕赤酵母</em&gt;。 electrocompetent酵母细胞的准备，与感兴趣的基因到改造的载体<em&gt; P。酵母</em&gt;和酵母DNA纯化。 Western blot分析和蛋白质纯化，建立在这种蛋白的表达协议的最后步骤。

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Research

JoVE Journal

Biology

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...