Expression of highly disulfide-bonded proteins in Pichia pastoris.
Expression of highly disulfide-bonded proteins in Pichia pastoris.
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DOI：
10.1016/s0969-2126(94)00103-0
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发表时间：
1994-11
期刊：
影响因子：
5.7
通讯作者：
C. E. White;N. Kempi;E. Komives
中科院分区：
文献类型：
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作者：
C. E. White;N. Kempi;E. Komives
大肠埃希菌不幸的是，E.大肠杆菌系统并不适用于所有蛋白质。例如，高度二硫键结合的蛋白质通常不能通过在E. coli的细胞质中，大肠杆菌是一个还原环境。因此，二硫键结合的蛋白质必须从E.大肠杆菌包涵体，或分泌到周质间隙。通常这两种策略都导致低产量。巴斯德毕赤酵母表达已成功地用于生产大量的二硫键结合的蛋白质，以及其他不能在细菌中表达的蛋白质。pastoris为E.大肠杆菌表达系统的原因。首先，蛋白质产生的P？巴斯德酵母通常正确折叠，因为酵母提供了与哺乳动物细胞中类似的细胞内折叠环境。其次，分泌蛋白到周围的介质中的P？可以容易地实现巴斯德酵母细胞的纯化，提供了纯化的实质性改进。第三，当在发酵罐中进行表达时，可以从多拷贝插入物获得每升培养物极高的蛋白质产量（通常100-500 mg 1-1）。第四，Pl巴斯德酵母可以在确定的培养基中生长，并且可以容易地获得15 N-或13 C-标记的蛋白。第五，巴斯德毕赤酵母的转化和生长不需要特殊的通风罩、培养箱或不同于用于E.杆菌最后，原生质球转化产生目的基因的多拷贝插入，大大提高了蛋白质的产量，并且很容易选择多拷贝插入transformants.We介绍了在这里的方法，用于原生质球转化的巴斯德毕赤酵母和获得（和选择）插入多拷贝的目的基因到醇氧化酶（AOX 1）基因的巴斯德毕赤酵母基因组。然后，我们提出了初步的数据，我们已经成功地表达在P？帕斯托里斯。
Escherichia coli. Unfortunately, E. coli systems are not suitable for all proteins. For example, highly disulfidebonded proteins usually cannot be made successfully by expression in E. coli because the cytoplasm of E. coli is a reducing environment. As a result of this, disulfidebonded proteins must be either refolded from E. coli inclusion bodies, or secreted into the periplasmic space. Typically both of these strategies result in low yields. Pichia pastoris yeast expression has been successfully utilized to produce large quantities of disulfide-bonded proteins, and other proteins that cannot be expressed in bacteria. pastoris provides an excellent alternative to E. coli expression systems for several reasons. Firstly, proteins produced in P? pastoris are usually folded correctly because yeast provide an intracellular folding environment similar to that in mammalian cells. Secondly, secretion of proteins into the medium surrounding the P? pastoris yeast cells can be easily accomplished, providing a substantial improvement in purification. Thirdly, extremely high yields of protein per liter of culture (typically 100-500 mg 1-1) can be obtained from multicopy inserts when expression is carried out in a fermenter. Fourthly, PI pastoris yeast can be grown in defined media and 15N-or 13 C-labeled proteins can be easily obtained. Fifthly, transformation and growth of P pastoris does not require special hoods, incubators or techniques other than those used for E. coli. Finally, spheroplast transformation produces multicopy insertion of the gene of interest, vastly increasing the yield of protein, and it is easy to select for multicopy insert transformants.We present here the methods for spheroplast transformation of P. pastoris yeast and for obtaining (and selecting for) insertion of multiple copies of the gene of interest into the alcohol oxidase (AOX1) gene of the P pastoris yeast genome. We then present preliminary data for production of a fragment of thrombomodulin which we have successfully expressed in P? pastoris.