title: Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli.
abstract: Enterokinase light chain (EKL) is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys (D(4)K) sequence and cleaves the C-terminal peptide bond of the lysine residue. The utility of EKL as a site-specific cleavage enzyme is hampered by sporadic cleavage at other sites than the canonical D(4)K recognition sequence. In order to produce more site-specific EKL, we have generated several EKL mutants in E. coli with substitutions at Tyr174 and Lys99 using PDI (protein disulfide isomerase) fusion system. Substitution of Tyr174 by basic residues confers higher specificity on EKL. The production of EKL with higher specificity could widen the utility of EKL as a site-specific cleavage enzyme to produce various recombinant proteins with therapeutic or industrial values.
authors.affiliation: Department of Bioinformatics and Life Science, Soongsil University, Seoul, 156-743, Korea.
authors.forename: Haarin
authors.initials: H
authors.lastname: Chun
authors.forename: Keehyoung
authors.initials: K
authors.lastname: Joo
authors.forename: Jooyoung
authors.initials: J
authors.lastname: Lee
authors.forename: Hang-Cheol
authors.initials: HC
authors.lastname: Shin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001709:Biotechnology; D002417:Cattle; D004765:Enteropeptidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D004765:Enteropeptidase