Widersten M
Biomedical Center, Uppsala University, Uppsala, S-751 23, Sweden.
Protein Expr Purif. 1998 Aug;13(3):389-95. doi: 10.1006/prep.1998.0913.
A system for heterologous expression in Escherichia coli of dehaloalkane dehalogenase Dh1A from Xanthobacter autotrophicus strain GJ10 is presented. The strategy involved overexpression of E. coli chaperonins GroEL/ES which facilitated the production of soluble Dh1A. When active-site mutant forms were constructed they could not to any detectable degree be expressed in a soluble state in the absence of overproduced GroEL/ES. However, with the described expression system, wild-type Dh1A as well as variant forms randomly mutated in the active-site residues Phe172 and Trp175 were reliably produced. An introduced C-terminal (His)5-tag provided an immunological handle as well as a site for metal ion coordination utilized in affinity chromatography for the purification of recombinant Dh1A. The purified His-tagged enzyme, Dh1A-5His, was confirmed to be catalytically fully active when measuring the dehalogenase activity with dichloroethane as substrate.
本文介绍了一种用于在大肠杆菌中异源表达自养黄色杆菌菌株GJ10的脱卤烷脱卤酶Dh1A的系统。该策略涉及大肠杆菌伴侣蛋白GroEL/ES的过表达，这有助于可溶性Dh1A的产生。当构建活性位点突变体形式时，在没有过量产生的GroEL/ES的情况下，它们无法以可检测的程度以可溶状态表达。然而，使用所述表达系统，可以可靠地产生野生型Dh1A以及在活性位点残基Phe172和Trp175处随机突变的变体形式。引入的C末端(His)5标签提供了一个免疫处理位点以及一个用于金属离子配位的位点，该位点用于亲和色谱法纯化重组Dh1A。当以二氯乙烷为底物测量脱卤酶活性时，纯化的His标签酶Dh1A-5His被证实具有完全催化活性。
