title: Functional expression and characterization of dipeptidyl peptidase IV from the black-bellied hornet Vespa basalis in Sf21 insect cells.
abstract: The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k(cat)/K(m) of rDPP-IV was determined to be in the range of 10-500 mM(-1)·S(-1) for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.
authors.affiliation: Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan.
authors.forename: Sheng-Kuo
authors.initials: SK
authors.lastname: Hsieh
authors.forename: Jason T C
authors.initials: JT
authors.lastname: Tzen
authors.forename: Tzong-Yuan
authors.initials: TY
authors.lastname: Wu
authors.forename: Ying-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Wei-Hung
authors.initials: WH
authors.lastname: Yang
authors.forename: Chun-Fa
authors.initials: CF
authors.lastname: Huang
authors.forename: Feng-Chia
authors.initials: FC
authors.lastname: Hsieh
authors.forename: Tzyy-Rong
authors.initials: TR
authors.lastname: Jinn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D014863:Wasps / Q000201:enzymology* / Q000235:genetics*
chemical_list: D018819:Dipeptidyl Peptidase 4