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Improved solubility of TEV protease by directed evolution.

Journal article
Authors Susanne van den Berg
Per-Åke Löfdahl
Torleif Härd
Helena Berglund
Published in Journal of biotechnology
Volume 121
Issue 3
Pages 291-8
ISSN 0168-1656
Publication year 2006
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 291-8
Language en
Links dx.doi.org/10.1016/j.jbiotec.2005.0...
Keywords Amino Acid Substitution, Catalytic Domain, Cell Separation, Cloning, Molecular, Directed Molecular Evolution, Endopeptidases, chemistry, genetics, Escherichia coli, genetics, metabolism, Escherichia coli Proteins, chemistry, Flow Cytometry, Gene Deletion, Genetic Techniques, Genetic Vectors, Green Fluorescent Proteins, metabolism, Histidine, chemistry, Protein Structure, Tertiary, Recombinant Fusion Proteins, metabolism, Recombination, Genetic, Solubility
Subject categories Medical and Health Sciences

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.

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