Cloning and Identification of a Novel P-II Class Snake Venom Metalloproteinase from Gloydius halys

Abstract

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.

Synergistic Effect of Proteinase Activity by Purification and Identification of Toxic Protease From Nemopilema nomurai

“…Many (Ami et al, 2017). Additionally, some metalloproteinases can be obtained by cDNA cloning and expression, such as rFIVa (Xu et al, 2006), Ahpfibrase (Zhang et al, 2010), and Jerdonitin (Zhu et al, 2010). Owing to the successful single protein purification, many SVMPs were studied in-depth in structures, functions, and mechanisms.…”

Snake Venom Metalloproteinases (SVMPs): A structure-function update

“…Continuous advancements in recombinant DNA technology have provided scientists with tools for the characterization of more protein structures. For instance, there was the successful cloning of the ahpfibrase from the venom of Gloydius halys ( Zhang et al, 2010 ). This P-II protein has 32 kDa and contains the canonical RGD-motif, which is responsible for platelet aggregation inhibition, besides its fibrinolytic activity ( Zhang et al, 2010 ).…”

“…For instance, there was the successful cloning of the ahpfibrase from the venom of Gloydius halys ( Zhang et al, 2010 ). This P-II protein has 32 kDa and contains the canonical RGD-motif, which is responsible for platelet aggregation inhibition, besides its fibrinolytic activity ( Zhang et al, 2010 ). In addition, Wang et al (2003) cloned the P-II agkistin from the venom of Agkistrodon halys and demonstrated that this protein of 57 kDa (as shown by Western blot profiling) inhibited ADP-induced platelet aggregation and induced apoptosis in Human Uterine Microvascular Endothelial Cells (HUMEC) ( Wang et al, 2003 ).…”

“… Dis Ba -01 8 Disintegrin Bothrops alternatus RGD α v β 3 – blocking effect and inhibition of angiogenesis in HUVEC Danilucci et al, 2019 , Ramos et al, 2008 13. Ahpfibrase 32 Metalloproteinase and disintegrin Gloydius halys RGD Fibrinolytic activity and inhibition of platelet aggregation Zhang et al (2010) 1 4 . Insularin 14 Disintegrin Bothrops insularis RGD Inhibition of endothelial cell adhesion Della-Casa et al (2011) 15.…”

Purification and characterization of platelet aggregation inhibitor from the Gloydius blomhoffii brevicaudus venom

“…Ahpfibrase (експресовано в культурі E. сoli) [50] *Цей рід дуже подібний до щитомордників роду Agkistrodon, розповсюджених в Америці, й обидві назви вживалися як синоніми. Але в 90-ті роки щитомордники Азії отримали правильну назву Gloydius.…”