Improved solubility of TEV protease by directed evolution.

• 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.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Biokatalys och enzymteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Biocatalysis and Enzyme Technology (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Annan industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Other Industrial Biotechnology (hsv//eng)

Nyckelord

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

TEV protease; Directed evolution; GFP; Solubility; FACS

Enzyme engineering

Other bioengineering

Publikations- och innehållstyp

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