Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox

Autor: Skala, Wolfgang, Goettig, Peter, Brandstetter, Hans
Rok vydání: 2013
Předmět:
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