title: Do-it-yourself histidine-tagged bovine enterokinase: a handy member of the protein engineer's toolbox.
abstract: 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.
authors.affiliation: Division of Structural Biology, Department of Molecular Biology, University of Salzburg, Billrothstraße 11, 5020 Salzburg, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Skala
authors.forename: Peter
authors.initials: P
authors.lastname: Goettig
authors.forename: Hans
authors.initials: H
authors.lastname: Brandstetter
mesh_terms: D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D002479:Inclusion Bodies / Q000201:enzymology; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D004765:Enteropeptidase
keywords: BENAC; Biotechnology; EK; Enteropeptidase; GST; IB; IEC; IMAC; In vitro folding; Inclusion bodies; KLK; SUMO; Serine proteases; TEV; benzamidine affinity chromatography; enterokinase; glutathione-S-transferase; inclusion body; ion exchange chromatography; kallikrein-related peptidase; mmobilized metal ion affinity chromatography; small ubiquitin-like modifier; tobacco etch virus; uPA; urokinase-type plasminogen activator