Expresión recombinante de la pictobina, una enzima similar a la trombina presente en el veneno de Bothrops pictus (jergón de costa)

Introduction. The recombinant expression of ophidian toxins has allowed to enhance their structural and functional study, especially in their biomedical applications. Pictobin is a thrombin-like enzyme from the venom of B. pictus, an endemic species of Peru, which has a high potential. Objective.to produce the recombinant version of Pictobin (rPictobin) in the eukaryotic model Pichia pastoris. Methods. We started with the commercial synthesis of the Pictobin nucleotide sequence (750 bp), which incorporates the EcoR I and Not I restriction sites. This sequence was amplified by PCR, inserted into the pPICZα-C vector and cloned into E. coli OneShot TOP10. Subsequently, the pPICZα-C–rPictobin plasmid was amplified for its transformation and expression in the Pichia pastorisGS115 system. Results. Expression was achieved after induction with 0.5% methanol for 7 days. The expression yield was 0.4 mg/L in P. pastoris. Additionally, SDS-PAGE analysis revealed that rPictobina has a molecular mass of 49 kDa, and showed amidolytic activity but no coagulant action on fibrinogen. ELISA and Western Blot analyses showed low recognition of the recombinant version by anti-B. pictusvenom antibodies. Conclusion. The production of the recombinant version of Pictobina was achieved, which has activity on synthetic substrate BApNA specific for serine proteases, allowing the first steps to be taken towards the heterologous production of this promising biomolecule