Expression, purification and molecular modeling of recombinant fibrinogenase [IV], a metalloproteinase from Deinakistrodon acutus venom.
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.
Xu
Li-Peng
LP
Department of Pharmacology, Sun Yat-sen University, Sun Yat-sen Medical School, 74 Zhongshan II Road, Guangzhou 510080, China.
Jiang
Wei-Jian
WJ
Ma
Tao
T
Qiu
Peng-Xin
PX
Hou
Jing
J
Huang
Yi-Jun
YJ
Chen
Jia-Shu
JS
Yan
Guang-Mei
GM
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
D012910:Snake Venoms; D005340:Fibrinogen; D045726:Metalloproteases