Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox
Autor: | Skala, Wolfgang, Goettig, Peter, Brandstetter, Hans |
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Rok vydání: | 2013 |
Předmět: |
EK
enterokinase KLK kallikrein-related peptidase Protein Folding Inclusion bodies IB inclusion body uPA urokinase-type plasminogen activator SUMO small ubiquitin-like modifier Protein Engineering Serine proteases Applied Microbiology and Biotechnology Article IMAC mmobilized metal ion affinity chromatography Recombinant Proteins TEV tobacco etch virus Enteropeptidase In vitro folding GST glutathione-S-transferase IEC ion exchange chromatography Escherichia coli Animals Cattle Histidine BENAC benzamidine affinity chromatography Cloning Molecular Biotechnology |
Zdroj: | Journal of Biotechnology |
ISSN: | 0168-1656 |
DOI: | 10.1016/j.jbiotec.2013.10.022 |
Popis: | Highlights • Histidine-tagged bovine enterokinase was refolded from bacterial inclusion bodies. • Refolding yields satisfy high demands of protein crystallography projects. • Enterokinase specifically cleaved artificial propeptides from target proteins. Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme. |
Databáze: | OpenAIRE |
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