[Expression, refolding and biological activity of recombinant type-I metalloproteinase acutolysin a from Agkistrodon acutus].
Type-I snake venom metalloproteinase acutolysin A gene was cloned into the prokaryotic expression vector pBAD/gIIIA and the resulting recombinant plasmid pDS was obtained. By the induction with 0.02% L-(+)-arabinose, the recombinant metalloproteinase was expressed in insoluble inclusion body in E. coli TOP10 and reached up to 5%--10% of total bacterial proteins. The recombinant metalloproteinase has an additional sequence of N-terminal 22 amino acids and C-terminal 8 amino acids (housing 6 histidines), both of which derived from the vector. The purified inclusion body was solubilized by 8 mol/L urea or 6 mol/L guanidine-HCl and the denatured soluble recombinant metalloproteinase was allowed to refold in vitro. Western blotting and ELISA obviously showed that the renatured recombinant metalloproteinase possessed strong immune reactivity very closely related to natural acutolysin A. Animal experiments showed that the refolded recombinant metalloproteinase had an obvious hemorrhagic activity. Except PMSF, 1 mmol/L EDTA, 1 mmol/L EGTA, and 3 mmol/L imidazole could inhibit the hemorrhagic activity of the recombinant and the natural metalloproteinases to different extent. Based on the investigations of others and our experimental results, the hemorrhagic mechanism of snake metalloproteinases was discussed.
Xiang
Kai-Jun
KJ
School of Life Science, University of Science and Technology of China, Hefei 230027, China. jliu@ustc.edu.cn
Yu
Hong-Xiu
HX
Zou
Chun-Sen
CS
Yuan
Pei-Hua
PH
Liu
Jing
J
D017836:Agkistrodon; D000818:Animals; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification
D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D008666:Metalloendopeptidases; C115407:acutolysin A