title: Production and purification of recombinant enteropeptidase expressed in an insect-baculovirus cell system.
abstract: Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT "A" baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-β-naphthylamide. The recombinant bovine enteropeptidase showed a K m value of 0.75 ± 0.02 mM with K cat 25 ± 1 s.
authors.affiliation: a Jawaharlal Nehru Technological University, Department of Biotechnology , Hyderabad , India.
authors.forename: Mahammad
authors.initials: M
authors.lastname: Azhar
authors.forename: R
authors.initials: R
authors.lastname: Somashekhar
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002417:Cattle; D002460:Cell Line; D004765:Enteropeptidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera
chemical_list: D011994:Recombinant Proteins; D004765:Enteropeptidase
keywords: Gly-(Asp) 4-Lys-β-naphthylamide; Ni-NTA affinity chromatography; bacmids; baculovirus; enteropeptidase; insect cells