Xie Ying, Han Xiao, Miao Yansong
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore.
Curr Protoc Mol Biol. 2018 Jul;123(1):e62. doi: 10.1002/cpmb.62. Epub 2018 Jun 5.
The expression and purification of recombinant proteins using bacterial vectors is a mature and preferred system to obtain folded and stable proteins. However, functional post-translational protein modifications, such as glycosylation or phosphorylation, can only be achieved using eukaryotic expression systems. In addition, insolubility is another challenge when using proteins expressed in Escherichia coli, such as certain intrinsically disordered proteins, which are more prone to aggregation than folded proteins. Eukaryotic protein expression systems, including human cells, baculovirus/insect cells, and yeast, have become indispensable for the production of functional eukaryotic proteins. This article describes a detailed protocol for performing cytosolic protein expression, protein purification, and protein characterization using the budding yeast Saccharomyces cerevisiae. The introduced protein expression and purification system in yeast are advantageous due to the low cost, high yield, high protein solubility, and minimal expertise required. © 2018 by John Wiley & Sons, Inc.
使用细菌载体表达和纯化重组蛋白是获得折叠且稳定蛋白的成熟且首选系统。然而，功能性的翻译后蛋白修饰，如糖基化或磷酸化，只能使用真核表达系统来实现。此外，当使用在大肠杆菌中表达的蛋白时，不溶性是另一个挑战，例如某些内在无序蛋白，它们比折叠蛋白更容易聚集。包括人类细胞、杆状病毒/昆虫细胞和酵母在内的真核蛋白表达系统已成为生产功能性真核蛋白不可或缺的工具。本文描述了使用酿酒酵母进行胞质蛋白表达及蛋白纯化和表征的详细方案。酵母中引入的蛋白表达和纯化系统具有成本低、产量高、蛋白溶解度高以及所需专业知识最少等优点。© 2018 约翰威立父子公司版权所有
