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

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 pr...

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Detalles Bibliográficos
Autores principales: Skala, Wolfgang, Goettig, Peter, Brandstetter, Hans
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier Science Publishers 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3863954/
https://www.ncbi.nlm.nih.gov/pubmed/24184090
http://dx.doi.org/10.1016/j.jbiotec.2013.10.022
Descripción
Sumario: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.