Expression,
Refolding and Biological Activity of Recombinant Type-I Metalloproteinase
Acutolysin A from Agkistrodon acutus
XIANG Kai-Jun, YU Hong-Xiu, ZOU Chun-Sen£¬ YUAN Pei-Hua, LIU Jing*
( School of Life Science, University of Science and Technology of China, Hefei
230027, China )
Abstract 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.
Key words snake venom metalloproteinase; expression;
refolding; hemorrhagic activity
*Corresponding author£º Tel, 86-551-3601437; Fax,
86-551-3601437; e-mail, [email protected]