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Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi, Ytbehandlingsteknik.
KTH, Skolan för teknikvetenskap (SCI).ORCID-id: 0000-0001-9423-0541
2019 (engelsk)Inngår i: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 400, nr 3, s. 405-415Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3C(pro)) towards improved proteolytic activity on the substrate LEVLFQ down arrow GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3C(pro) library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

sted, utgiver, år, opplag, sider
WALTER DE GRUYTER GMBH , 2019. Vol. 400, nr 3, s. 405-415
Emneord [en]
Coxsackievirus B3, FACS, GFP-fusion, intracellular assay, protease engineering, protease substrate profiling
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-245129DOI: 10.1515/hsz-2018-0362ISI: 000458628400014PubMedID: 30521472Scopus ID: 2-s2.0-85058219114OAI: oai:DiVA.org:kth-245129DiVA, id: diva2:1296057
Merknad

QC 20190313

Tilgjengelig fra: 2019-03-13 Laget: 2019-03-13 Sist oppdatert: 2019-03-18bibliografisk kontrollert

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Meister, SebastianHendrikse, NatalieLöfblom, John

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