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Improved solubility...
Improved solubility of TEV protease by directed evolution.
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- van den Berg, Susanne (author)
- Karolinska Institutet
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- Löfdahl, Per-Åke, 1959- (author)
- KTH,Fysik,Structural Biochemistry
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- Härd, Torleif, 1959 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi,Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
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Berglund, Helena (author)
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(creator_code:org_t)
- Amsterdam : Elsevier BV, 2006
- 2006
- English.
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In: Journal of biotechnology. - Amsterdam : Elsevier BV. - 0168-1656 .- 1873-4863. ; 121:3, s. 291-8
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Abstract
Subject headings
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- 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.
Subject headings
- 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)
Keyword
- 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
Publication and Content Type
- ref (subject category)
- art (subject category)
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