Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self‐cleavage exhibits enhanced enzymatic activity
| Autor: | Sooim Shin, Moonsung Choi, Deog-Young Choi, Heejin Nam, Beom Jeung Hwang |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Proteases substrate specificity QH301-705.5 medicine.medical_treatment Potyvirus Mutant inclusion body tobacco etch virus protease General Biochemistry Genetics and Molecular Biology self‐inactivation Viral Proteins 03 medical and health sciences 0302 clinical medicine Catalytic Domain enzyme kinetics Endopeptidases Escherichia coli TEV protease medicine Amino Acid Sequence Enzyme kinetics Biology (General) Peptide sequence Research Articles Inclusion Bodies chemistry.chemical_classification Protease biology Chemistry Tobacco etch virus multiple mutations biology.organism_classification Enzyme Activation Kinetics 030104 developmental biology Enzyme Solubility Biochemistry 030220 oncology & carcinogenesis Mutation Research Article Plasmids |
| Zdroj: | FEBS Open Bio, Vol 10, Iss 4, Pp 619-626 (2020) FEBS Open Bio |
| ISSN: | 2211-5463 |
| Popis: | Tobacco etch virus (TEV) protease is a 27‐kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. Despite its substrate specificity, its use is limited by its autoinactivation through self‐cleavage and poor solubility during purification. It was previously reported that T17S/N68D/I77V mutations improve the solubility and yield of TEV protease and S219 mutations provide protection against self‐cleavage. In this study, we isolated TEV proteases with S219N and S219V mutations in the background of T17S, N68D, and I77V without the inclusion body, and measured their enzyme kinetics. The k cat of two isolated S219N and S219V mutants in the background of T17S, N68D, and I77V mutations was highly increased compared to that of the control, and S219N was twofold faster than S219V without K m change. This result indicates that combination of these mutations can further enhance TEV activity. The use of tobacco etch virus (TEV) protease is limited by its poor solubility and self‐inactivation. We analyzed the effect of quadruple mutations (T17S, N68D, I77V, and S219X), which affect solubility and inhibit autoinactivation in TEV protease. In a background of T17S, N68D, and I77V mutations, soluble S219N mutant TEV protease serves as a more efficient catalyst than soluble S219V mutant. |
| Databáze: | OpenAIRE |
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