Isolation, characterization, and expression of the Montivipera raddei (Caucasus viper) venom metalloprotease-like protein.

Background and purpose: Snake venom contains numerous proteolytic enzymes, including metalloproteases. This study aimed to isolate, characterize the metalloproteinase from Montivipera raddei venom and insert its cDNA into a host cell genome for expressing the protein's potential use as a coagulation factor.

Experimental approach: Initially, M. raddei venom was analyzed using SDS-PAGE and ion-exchange chromatography. All purified fractions were assessed using a prothrombin time (PT) assay. Immunoblot analysis and MALDI-TOF/TOF MS spectrometry confirmed the metalloproteinase active domain in the obtained fraction. All of the confirmations were studied using the I-TASSER server. To produce the recombinant enzyme, metalloproteinase cDNA was isolated from the total RNA of the Caucasus viper venom gland tissue and cloned into pEX-A2-H plasmid, containing homologous sequences with CHO-S cell genome. Also, it was confirmed by western blotting and PT test.

Findings/results: Two fractions from M. raddei venom had PT values of 6 and 21 s, respectively. We confirmed the presence of a 25-KDa procoagulant metalloprotease (Met). The results indicated successful expression of the Met protein in the recombinant CHO-S cells. The Met protein was structurally similar to the target in the PDB ID. 2e3x, regulatory subunit of the blood coagulation factor X- and IX-activating enzyme. The Met protein also contained a similar binding domain with 60 amino acids, comparable to those in E. carinatus. and E. pyramidum leakeyi metalloproteinases.

Conclusion and implications: The Met protein may be a potent candidate recombinant coagulant drug. Remarkably, the thrombin time for this protein was 3 s.