Cloning and identification of a novel P-II class snake venom metalloproteinase from Gloydius halys.
Ahpfibrase was a new snake venom metalloproteinase (SVMP) which was cloned from Gloydius halys. The cDNA sequence with 1,891 base pairs encodes an open reading frame of 477 amino acids which includes a 17 amino acid signal peptide, plus a 171 amino acid segment of zymogen-like propeptide, a metalloproteinase domain of 200 amino acids, a spacer of 16 amino acids, and a disintegrin-like peptide of 73 amino acids. The metalloproteinase domain contained a conserved signature zinc-binding motif HEXXHXXGXXH in the catalytic region and a methionine-turn CIM. To determine the activity of ahpfibrase, the coding region including both the metalloproteinase domain and disintegrin region was amplified by PCR, inserted into the pET25b(+) vector, and expressed in Escherichia coli. The recombinant protein was recovered from inclusion bodies with 8 M urea and refolding was performed by fed-batch dilution method, and purified recombinant ahpfibrase showed the fibrinolytic activity and platelet aggregation-inhibition ability.
Zhang
Shou-Tao
ST
Department of Bioengineering, Zhengzhou University, Zhengzhou, Henan 450001, China. zhangst@zzu.edu.cn
Lu
Ping
P
Qin
Yun-Fei
YF
Chen
San-Jun
SJ
Guo
Ai-Guang
AG
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002364:Caseins / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D045726:Metalloproteases / Q000737:chemistry / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology; D017510:Protein Folding / Q000187:drug effects; D017422:Sequence Analysis, DNA; D012910:Snake Venoms / Q000201:enzymology*; D013696:Temperature; D017819:Viperidae / Q000378:metabolism*
D002364:Caseins; D002413:Cations, Divalent; D011480:Protease Inhibitors; D012910:Snake Venoms; C026018:azocasein; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D045726:Metalloproteases