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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.

SUBMITTER: Skala W

PROVIDER: S-EPMC3863954 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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

Skala Wolfgang W Goettig Peter P Brandstetter Hans H

Journal of biotechnology 20131030 4

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 ligh ...[more]

PMID: 24184090

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