Heterologous expression in Escherichia coli of soluble active-site random mutants of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 by coexpression of molecular chaperonins GroEL/ES.
Heterologous expression in Escherichia coli of soluble active-site random mutants of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 by coexpression of molecular chaperonins GroEL/ES.
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通过共表达分子伴侣 GroEL/ES，在大肠杆菌中异源表达来自自养黄杆菌 GJ10 的卤代烷脱卤酶的可溶性活性位点随机突变体。
DOI：
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发表时间：
1998
影响因子：
1.6
通讯作者：
M. Widersten
中科院分区：
文献类型：
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作者：
M. Widersten
提出了一种在大肠杆菌中异源表达来自自养黄杆菌菌株 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 的过度表达，从而促进可溶性 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，它们不能以任何可检测的程度以可溶状态表达。然而，利用所描述的表达系统，可以可靠地产生野生型 Dh1A 以及活性位点残基 Phe172 和 Trp175 中随机突变的变体形式。引入的 C 端 (His)5 标签提供了免疫学手柄以及用于亲和层析纯化重组 Dh1A 的金属离子配位位点。当以二氯乙烷为底物测量脱卤素酶活性时，纯化的 His 标记酶 Dh1A-5His 被证实具有完全催化活性。
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.