title: Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin.
abstract: Echistatin (Ech) is a potent inhibitor of integrins and was isolated from snake Echis carinatus. To facilitate the study on the molecular determinants of integrin-ligand interactions, we expressed recombinant Ech and its mutants in the Pichia pastoris (P. pastoris) expression system and purified them to homogeneity with the yields of 2-7 mg/L. Ech produced in P. pastoris inhibited platelet aggregation with the IC(50) value of 210.5 nM. The sequential assignment and structure analysis of recombinant Ech were obtained by 2D and 3D (15)N-edited NMR spectra. These data suggests that Ech produced in P. pastoris retained its function and native fold. The results presented here provide the evidences that four disulfide-bonded peptide inhibitor of integrin, Ech, can be expressed in P. pastoris with correct fold and high yield. Platelet aggregation analysis of Ech mutants showed that the length of C-terminus and the K45 residue of Ech are important for interacting with integrin αIIbβ3. We also found that recombinant Ech can inhibit the migration of human A375 melanoma cell. These findings may serve as the basis for understanding the activities of Ech.
authors.affiliation: Institute of Basic Medical Sciences and Department of Biochemistry, National Cheng Kung University Medical College, Tainan 701, Taiwan.
authors.forename: Yi-Chun
authors.initials: YC
authors.lastname: Chen
authors.forename: Chun-Ho
authors.initials: CH
authors.lastname: Cheng
authors.forename: Jia-Hau
authors.initials: JH
authors.lastname: Shiu
authors.forename: Yao-Tsung
authors.initials: YT
authors.lastname: Chang
authors.forename: Yung-Sheng
authors.initials: YS
authors.lastname: Chang
authors.forename: Chun-Hau
authors.initials: CH
authors.lastname: Huang
authors.forename: Jenq-Chang
authors.initials: JC
authors.lastname: Lee
authors.forename: Woei-Jer
authors.initials: WJ
authors.lastname: Chuang
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D019483:Disintegrins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D010843:Pichia / Q000235:genetics*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D014757:Viper Venoms / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D019483:Disintegrins; D036341:Intercellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D014757:Viper Venoms; C057986:echistatin; C047981:arginyl-glycyl-aspartic acid