Back to Search Start Over

Improved solubility of TEV protease by directed evolution

Authors :
Per-Åke Löfdahl
Torleif Härd
Helena Berglund
Susanne van den Berg
Source :
Journal of Biotechnology. 121:291-298
Publication Year :
2006
Publisher :
Elsevier BV, 2006.

Abstract

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

Details

ISSN :
01681656
Volume :
121
Database :
OpenAIRE
Journal :
Journal of Biotechnology
Accession number :
edsair.doi.dedup.....080a6945db2d20ec1ba5ca9443ced3eb
Full Text :
https://doi.org/10.1016/j.jbiotec.2005.08.006