Produção heteróloga de uma protease cisteínica de Calotropis procera (Apocynaceae) e avaliação da sua funcionalidade

Abstract

Cysteine proteases (EC 3.4.22.−) have a catalytic mechanism that involves the thiol group of a cysteine residue with a nucleophilic character, forming a catalytic dyad (Cys/His). Within this group, several enzymes have been identified, such as those present in the latex of Calotropis procera. The CpCP3 protease from C. procera latex had not yet been heterologously obtained in its active form. This study aimed to express the cysteine peptidase CpCP3 in Escherichia coli and demonstrate its functionality. To this end, the peptidase sequence was expressed fused to different solubility tags: thioredoxin A (TrxA), glutathione S-transferase (GST), and the transcription termination/antitermination protein (NusA). When produced in E. coli BL21(DE3), the fusion proteins exhibited molecular masses of 42.9 kDa (TrxA-CpCP3), 57.7 kDa (GST-CpCP3), and 85.9 kDa (NusA-CpCP3), as analyzed by SDS-PAGE. Pilot inductions revealed that the fusion proteins, after cell lysis in standard buffer (50 mM Tris-HCl; 0.5 M NaCl; 2 mM EDTA; 10% glycerol at pH 8.0), accumulated as insoluble inclusion bodies. Attempts to solubilize the recombinant proteins by incorporating the CTAB detergent into the lysis buffer at concentrations ranging from 6% to 10% did not improve solubility. Similarly, solubilizing the pellets with urea solutions (2 M to 8 M, at pH 8.0) was ineffective. However, when 8 M urea at pH 12.5 was incorporated into the lysis buffer, SDS-PAGE analysis showed significant solubilization of the fusion proteins. Despite the various methodological approaches employed, protein purification was not achieved, even using immobilized metal affinity chromatography (IMAC) with nickel and cobalt, ion exchange chromatography (DEAE-Sepharose), and size-exclusion chromatography. Enzyme activity assays with protein extracts showed positive results. The protein fused to the TrxA tag exhibited minimal activity, evidenced by the in vitro degradation of BSA. In contrast, the protein fused to the NusA tag showed significantly higher activity, being able to degrade gelatin in a zymogram and degrade BSA in vitro. In the three-dimensional modeling, the native structure of the protein exhibited an optimal conformation, showing high similarity to crystallized cysteine proteases (procerain, procerain B, and papain). In molecular docking, the catalytic site of the protease was observed to interact significantly with the ligands, being inhibited by E-64 and effectively cleaving the BANA substrate.