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Improved solubility of TEV protease by directed evolution
Susanne van den Berg1, Per-Ake Löfdahl, Torleif Härd
1Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE 171 77 Stockholm, Sweden.
Journal of Biotechnology
|September 10, 2005
Summary
Directed evolution improved the solubility of tobacco etch virus (TEV) protease, a key enzyme for protein cleavage. A new mutant, TEV(SH), shows a five-fold increase in yield with retained activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Tobacco etch virus (TEV) protease is highly efficient and specific for cleaving recombinant fusion proteins.
- Low protein solubility during expression in E. coli limits the production yield of TEV protease.
Purpose of the Study:
- To enhance the yield of soluble TEV protease through directed evolution.
- To identify TEV protease mutants with improved solubility and retained enzymatic activity.
Main Methods:
- Directed evolution using error-prone PCR and gene shuffling to create mutant TEV protease libraries.
- Gateway cloning system for efficient library manipulation.
- Fluorescence-based in vivo screening using a C-terminal GFP fusion to identify soluble variants.
- Expression, purification, and activity testing of promising mutants using a C-terminal histidine tag.
Main Results:
- Identified a TEV protease mutant, designated TEV(SH), with three amino acid substitutions.
- TEV(SH) exhibited a five-fold increase in the yield of purified protease compared to the wild-type.
- The enzymatic activity of the TEV(SH) mutant was retained.
Conclusions:
- Directed evolution is an effective strategy for improving the solubility and production yield of TEV protease.
- The TEV(SH) mutant offers a significant advancement for applications requiring large quantities of active TEV protease.
- This work provides a valuable tool for recombinant protein processing in biotechnology and research.