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.
van den Berg
Susanne
S
Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE 171 77 Stockholm, Sweden.
Löfdahl
Per-Ake
PA
Härd
Torleif
T
Berglund
Helena
H
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D002469:Cell Separation; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D005434:Flow Cytometry; D017353:Gene Deletion; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012995:Solubility
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease