RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes. Usually, the sacrifice of snakes is necessary for venom gland extraction to identify protein-coding transcripts; however, the venom can be used as a source of transcripts. Although there are methods for obtaining RNA from venom, no comparative analysis has been conducted in the Bothrops genus. In the present study, we compared four commercial methods for RNA purification and cDNA synthesis from venom (liquid, lyophilized, or long-term storage) of four clinically relevant species of Peruvian Bothrops. Our results show that the TRIzol method presents the highest yield of RNA purified from venom (59 ± 11 ng/100 µL or 10 mg). The SuperScript First-Strand Synthesis System kit produced high amounts of cDNA (3.2 ± 1.2 ng cDNA/ng RNA), and the highest value was from combination with the Dynabeads mRNA DIRECT kit (4.8 ± 2.0 ng cDNA/ng RNA). The utility of cDNA was demonstrated with the amplification of six relevant toxins: thrombin-like enzymes, P-I and P-III metalloproteinases, acid and basic phospholipases A2, and disintegrins. To our knowledge, this is the first comparative study of RNA purification and cDNA synthesis methodologies from Bothrops genus venom.

Lys49 phospholipase A2 (Lys49-PLA₂) homologues are catalytically inactive snake venom toxins with diverse biological activities, but their functional characterization requires recombinant production strategies to overcome the limitations inherent to native venom-derived proteins. Here, we produced recombinant BaMtx (rBaMtx), a Lys49-PLA₂ homologue from Bothrops atrox, in the Pichia pastoris KM71 expression system and evaluated whether its biochemical and biological properties were preserved. rBaMtx was secreted into the culture medium, purified by cation-exchange chromatography, and characterized by SDS-PAGE, Western blotting, RP-HPLC (85.9% purity), and MALDI-TOF mass spectrometry, confirming a molecular mass of 13,821 Da. Despite lacking PLA₂ activity, rBaMtx retained pronounced myotoxicity in vivo, inducing dose-dependent plasma creatine kinase release and skeletal muscle damage comparable to or greater than native BaMtx. In vitro, rBaMtx reduced viability of murine 4T1 breast carcinoma cells (IC₅₀ = 60.94 µg/mL), altered cell morphology, modestly increased IL-1β release, and produced context-dependent effects in paired human tumor and non-tumor cell models, differing from native BaMtx and crude venom. These findings establish rBaMtx as a reproducible model for investigating the mechanisms and biomedical potential of catalytically inactive Lys49-PLA₂ homologues.