Large-scale purification of a stable form of recombinant tobacco etch virus protease

被引：104

作者：

Lucast, LJ ^{[1
]}

Batey, RT ^{[1
]}

Doudna, JA ^{[1
]}

机构：

[1] Yale Univ, Dept Mol Biophys & Biochem, Howard Hughes Med Inst, New Haven, CT 06511 USA

来源：

BIOTECHNIQUES | 2001年 / 30卷 / 03期

关键词：

D O I：

10.2144/01303st06

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Tobacco etch virus NIa proteinase (NIa-Pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro. We have designed a mutant NIa-Pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pare, active and stable over time. Histidine-tagged forms of both wild-type and mutant NIa-Pro were overexpressed in E. coli under conditions in which greater than 95% of the protease was in the insoluble fraction after cell lysis. An inclusion body preparation followed by denaturing purification over a single affinity column and protein renaturation yields greater than 12.5 mg enzyme per lifer of bacterial cell culture. NIa-Pro purified according to this protocol has been used for quantitative removal of fusion domains from a variety of proteins prepared for crystallization and biochemical analysis.

引用

页码：544 / +

页数：5

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