Expression, purification and molecular modeling of recombinant fibrinogenase [IV], a metalloproteinase from Deinakistrodon acutus venom

• DOI: 10.1016/j.toxicon.2005.11.005
• Publication Date: 2005-12-29T12:13:10Z
• Authors: Li-Peng Xu; Wei-Jian Jiang; Tao Ma; Peng-Xin Qiu; Jing Hou; Yi-Jun Huang; Jia-Shu Chen; Guang-Mei Yan
• Keywords: Models, Molecular; 0301 basic medicine; Sequence Homology, Amino Acid; Protein Conformation; Temperature; Fibrinogen; Gene Expression; Snakes; Hydrogen-Ion Concentration; 03 medical and health sciences; Enzyme Stability; Metalloproteases; Animals; Amino Acid Sequence; Sequence Alignment; Snake Venoms

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.