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Improved solubility of TEV protease by directed evolution
- 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.
- Subjects :
- Gateway Technology
Recombinant Fusion Proteins
medicine.medical_treatment
Genetic Vectors
Green Fluorescent Proteins
Mutant
Bioengineering
Cell Separation
Applied Microbiology and Biotechnology
Catalytic Domain
Endopeptidases
Escherichia coli
TEV protease
medicine
Histidine
Cloning, Molecular
Recombination, Genetic
Tandem affinity purification
Protease
biology
Tobacco etch virus
Escherichia coli Proteins
General Medicine
Flow Cytometry
biology.organism_classification
Directed evolution
Molecular biology
Fusion protein
Protein Structure, Tertiary
Amino Acid Substitution
Genetic Techniques
Solubility
Biochemistry
Directed Molecular Evolution
Gene Deletion
Biotechnology
Subjects
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