Esterase 2 from Alicyclobacillus acidocaldarius as a reporter and affinity tag for expression and single step purification of polypeptides

被引：11

作者：

Huang, Yiwei ^{[1
]}

Humenik, Martin ^{[1
]}

Sprinzl, Mathias ^{[1
]}

机构：

[1] Univ Bayreuth, Biochim Lab, D-95440 Bayreuth, Germany

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2007年 / 54卷 / 01期

关键词：

esterase; reporter enzyme; affinity tag; trifluoromethyl-alkyl-ketone;

D O I：

10.1016/j.pep.2007.02.005

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A novel dual function (reporter and affinity) tag system has been developed. Expression vectors have been constructed to express polypeptides in Escherichia coli cells as C-terminal fusions with esterase 2, a 34-kDa protein from Alicyclobacillits acidocaldarius. Presence of esterase allows to monitor the expression of fusion proteins spectrophotornetrically or by activity staining in the polyacrylamide gels. The fusion proteins can be purified from crude bacterial extracts under non-denaturing conditions by one step affinity chromatography on Sepharose CL-6B immobilized trifluoromethyl-alkyl-ketone. The esterase carrier can be cleaved from fusion proteins by digestion with amino acid sequence-specific proteases blood coagulation factor Xa. The system has been used successfully for the expression and purification of polypeptides from different prokaryotic and eukaryotic organisms. (c) 2007 Elsevier Inc. All rights reserved.

引用

页码：94 / 100

页数：7

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