title: Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA.
abstract: Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage reagent. Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EKL) was identified, characterized, and transiently expressed in mammalian COS cells. We report here the production of EKL in Escherichia coli by a novel secretory expression system that utilizes E. coli DsbA protein as an N-terminal fusion partner. The EKL cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site. Active, processed recombinant EKL (rEKL) was generated from this fusion protein via an autocatalytic cleavage reaction. The enzymatic properties of the bacterially produced rEKL were indistinguishable from the previously described COS-derived enzyme. Both forms of rEKL were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena. Interestingly, rEKL activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)4-Lys recognition sequence.
authors.affiliation: Genetics Institute, Inc, Cambridge, MA 02140, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Collins-Racie
authors.forename: J M
authors.initials: JM
authors.lastname: McColgan
authors.forename: K L
authors.initials: KL
authors.lastname: Grant
authors.forename: E A
authors.initials: EA
authors.lastname: DiBlasio-Smith
authors.forename: J M
authors.initials: JM
authors.lastname: McCoy
authors.forename: E R
authors.initials: ER
authors.lastname: LaVallie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002384:Catalysis; D002417:Cattle; D003593:Cytoplasm / Q000201:enzymology; D019281:Dimerization; D004765:Enteropeptidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004765:Enteropeptidase; D019704:Protein Disulfide-Isomerases