Engineering of TEV protease variants with redesigned substrate specificity.
GFP-fusion
TEV protease
amyloid betas
intracellular assay
protease engineering
substrate specificity
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
Nov 2023
Nov 2023
Historique:
revised:
16
06
2023
received:
14
12
2022
accepted:
07
07
2023
medline:
13
11
2023
pubmed:
14
7
2023
entrez:
14
7
2023
Statut:
ppublish
Résumé
Due to their ability to catalytically cleave proteins and peptides, proteases present unique opportunities for the use in industrial, biotechnological, and therapeutic applications. Engineered proteases with redesigned substrate specificities have the potential to expand the scope of practical applications of this enzyme class. We here apply a combinatorial protease engineering-based screening method that links proteolytic activity to the solubility and correct folding of a fluorescent reporter protein to redesign the substrate specificity of tobacco etch virus (TEV) protease. The target substrate EKLVFQA differs at three out of seven positions from the TEV consensus substrate sequence. Flow cytometric sorting of a semi-rational TEV protease library, consisting of focused mutations of the substrate binding pockets as well as random mutations throughout the enzyme, led to the enrichment of a set of protease variants that recognize and cleave the novel target substrate.
Identifiants
pubmed: 37448316
doi: 10.1002/biot.202200625
doi:
Substances chimiques
TEV protease
EC 3.4.-
Endopeptidases
EC 3.4.-
Peptide Hydrolases
EC 3.4.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2200625Subventions
Organisme : Swedish Brain Foundation
ID : FO2018-0094
Organisme : Swedish Brain Foundation
ID : FO2021-0407
Organisme : Swedish Brain Foundation
ID : FO2022-0253
Organisme : Swedish Brain Foundation
ID : FO2023-0400
Organisme : Swedish Brain Foundation
ID : FO2023-0141
Organisme : Knut and Alice Wallenberg Foundation
ID : KAW 2019.0341
Organisme : Knut and Alice Wallenberg Foundation
ID : KAW 2021.0197
Organisme : Tussilago Foundation
ID : FL-0002.025.551-7
Organisme : Schörling Family Foundation
Organisme : Swedish Cancer Society
ID : CAN 20 1090 PjF
Organisme : Swedish Cancer Society
ID : 19 0101 Pj01H
Organisme : Swedish Cancer Society
ID : SS 22 2023 Pj01H
Organisme : Swedish Research Council
ID : 2019-05115
Organisme : Swedish Agency for Innovation VINNOVA
ID : 2019/00104
Organisme : Swedish Agency for Innovation VINNOVA
ID : 2017/02105
Informations de copyright
© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.
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