Nigi Irene, Fairall Louise, Schwabe John W R
Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.
Curr Protoc Protein Sci. 2017 Nov 1;90:5.28.1-5.28.16. doi: 10.1002/cpps.44.
Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies. Mammalian expression systems can often produce natively folded proteins, sometimes with native post-translational modifications. However, such expression systems are underutilized due to the perception that they are costly, technically challenging and result in limited protein yields. In fact, HEK 293F cells are straightforward to grow, transfect with high efficiency and often produce significant yields of recombinant proteins. In this unit, we describe a method to express and purify milligram quantities of a human protein complex from HEK 293F cells grown in suspension transiently transfected with the appropriate plasmids. © 2017 by John Wiley & Sons, Inc.
原核表达系统已被广泛用于表达蛋白质以进行结构研究。这类表达系统具有经济、直接和快速的优点。然而，对于许多真核蛋白质，尤其是蛋白质复合物，细菌表达系统无法产生大量的可溶性蛋白质。这可能是由于未能有效转录/翻译所需蛋白质，也可能是由于形成了称为包涵体的不溶性聚集体。哺乳动物表达系统通常可以产生天然折叠的蛋白质，有时还具有天然的翻译后修饰。然而，由于人们认为这类表达系统成本高、技术难度大且蛋白质产量有限，因此未得到充分利用。事实上，HEK 293F细胞易于培养，转染效率高，并且通常能产生大量的重组蛋白质。在本单元中，我们描述了一种从瞬时转染了适当质粒的悬浮培养的HEK 293F细胞中表达和纯化毫克量人蛋白质复合物的方法。© 2017约翰威立父子公司版权所有
