title: Expression, purification and molecular modeling of recombinant fibrinogenase [IV], a metalloproteinase from Deinakistrodon acutus venom.
abstract: A novel metalloproteinase, recombinant fibrinogenase IV (rFIV(a)), was expressed and purified from Deinakistrodon acutus venom. It was a single chain protein with an apparent molecular weight 27 kDa and an isoeletric point of pH 7.1. RFIV(a) cleaved preferentially the Aalpha-chain and also cleaved Bbeta, gamma-chains of fibrinogen when the incubation time was prolonged. The proteolytic activity was inhibited by EDTA, l-cysteine, and DTT, indicating rFIV(a) was a metalloproteinase requiring disulfide bonds for its activity. It kept above 85% of the initial activity from pH 4.5-11, showed an equal maximum activity at the temperature range from 30 to 50 degrees C, and was inactivated by Zn2+, Cu2+ and Cd2+. Homology modeling of rFIV(a) showed that two highly conserved disulfide bonds (Cys159-Cys164 and Cys117-Cys197) was maintained from its structure, and it exhibited the characteristic conserved motif H142E143XXH146XXGXXH152, whose three histidine residues were involved in binding of the catalytically essential zinc ion. This work demonstrates the expression, purification and characterization of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from D. acutus venom.
authors.affiliation: Department of Pharmacology, Sun Yat-sen University, Sun Yat-sen Medical School, 74 Zhongshan II Road, Guangzhou 510080, China.
authors.forename: Li-Peng
authors.initials: LP
authors.lastname: Xu
authors.forename: Wei-Jian
authors.initials: WJ
authors.lastname: Jiang
authors.forename: Tao
authors.initials: T
authors.lastname: Ma
authors.forename: Peng-Xin
authors.initials: PX
authors.lastname: Qiu
authors.forename: Jing
authors.initials: J
authors.lastname: Hou
authors.forename: Yi-Jun
authors.initials: YJ
authors.lastname: Huang
authors.forename: Jia-Shu
authors.initials: JS
authors.lastname: Chen
authors.forename: Guang-Mei
authors.initials: GM
authors.lastname: Yan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012910:Snake Venoms / Q000201:enzymology*; D012911:Snakes; D013696:Temperature
chemical_list: D012910:Snake Venoms; D005340:Fibrinogen; D045726:Metalloproteases