Goh Kian Mau, Liew Kok Jun, Chai Kian Piaw, Illias Rosli Md
Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
Methods Mol Biol. 2017;1498:385-396. doi: 10.1007/978-1-4939-6472-7_27.
Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
蛋白质工程是探究蛋白质结构与功能关系的一种非常有用的工具。具体而言，定点诱变的蛋白质能够为蛋白质的结构、结合及催化机制提供有益的见解，尤其是与结晶技术相结合时。在本章中，我们描述了两种对任何蛋白质编码序列进行定点诱变的方法，即大引物PCR和重叠延伸PCR（OE-PCR）。我们以这两种定点诱变方法如何增强来自芽孢杆菌G1菌株的环糊精葡萄糖基转移酶（CGTase）的功能为例进行说明。
