Expression and Purification of a Plasminogen Activator from the Venom of the Snake Lachesis muta (rLm-PA)

In Brazil, Lachesis envenomations, although less frequent, are among the most lethal. Their venom contains serine proteases (SVSPs) with fibrinolytic properties, including plasminogen activators with therapeutic potential. Given the narrow therapeutic window, PAI-1 susceptibility, and bleeding risk of current thrombolytics, recombinant expression of these enzymes offers a promising route to new biotechnological agents. This study aimed to express, purify and functionally characterize, in mammalian cells, a recombinant plasminogen activator from Lachesis muta venom (rLm-PA). Analysis of the L. muta venom gland transcriptome identified a transcript encoding a SVSP homologous to the plasminogen activator LV-PA. In silico analyses showed high identity with LV-PA and TSV-PA, conservation of the catalytic triad (His-Asp-Ser) and absence of N-terminal basic residues associated with PAI-1 inhibition, suggesting potential for sustained fibrinolytic activity. The sequence was optimized for eukaryotic systems and synthesized in the pcDNA3.4 vector for use in CHO cells. Cells were transfected with Lipofectamine™ 3000, and rLm-PA expression was evaluated from 24 to 96 h by SDS-PAGE and Western blot. Stable cell lines were then generated by electroporation, selected with G418 and analyzed by the same techniques. rLm-PA was purified by nickel affinity chromatography and tested for plasminogen activation, using the chromogenic substrate S-2251, and for degradation of plasminogen-free human fibrinogen. rLm-PA was expressed and secreted by CHO cells, with a progressive increase up to 96 h, and the G418-selected cell line maintained production of the active protein. The purified fraction showed a band of approximately 30 kDa, as expected for rLm-PA, and another of 66 kDa, probably serum albumin. The 30 kDa band was recognized by anti-HisTag and anti-LV-PA antibodies and by anti-Lachesis serum, but not by anti-Bothrops serum. Preliminary results show that rLm-PA converts human plasminogen into active plasmin, confirming its biological activity, and does not degrade human fibrinogen, which may reduce the risk of systemic bleeding. In conclusion, rLm-PA retains plasminogen-activating ability. To our knowledge, this is the first SVSP with plasminogen-activating activity expressed in mammalian cells, advancing the characterization of a candidate thrombolytic agent.