Large-Scale Purification of a Stable Form of Recombinant Tobacco Etch Virus Protease

Citations Over TimeTop 10% of 2001 papers

Abstract

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 pure, 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 liter 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.

Related Papers

• → New donor vector for generation of histidine-tagged fusion proteins using the Gateway Cloning System(2003)18 cited
• → Regulation of Histidine Uptake by Specific Feedback Inhibition of Two Histidine Permeases in Saccharomyces cerevisiae(1970)141 cited
• → Effect of Induction of Histidase on Histidine Metabolism in vivo(1979)14 cited
• → Presence and measurement of sample histidine in the ames test: Quantification and possible elimination of a source of false‐positive mutagenicity test results(1987)10 cited
• → Histidine transport into isolated animal cells(1968)26 cited