Expressão heteróloga, solubilização e purificação de uma osmotina antifúngica de Calotropis procera

Abstract

In the preliminary stage of this work, an osmotin was purified from the latex of Calotropis procera, called CpOsm. This protein exhibits strong activity against phytopathogenic fungi, presenting great biotechnological potential in the development of antifungals. However, the application of recombinant DNA technology for the production and recovery of rCpOsm is still a challenge. Thus, the present work aimed to establish an optimized protocol for the expression and purification of this osmotin. For this, CpOsm was expressed in E. coli BL21(DE3) cells fused to the thioredoxin (Trx) fusion tag. The inclusion bodies, containing Trx-CpOsm, were efficiently solubilized using 1% (w/v) cetyltrimethylammonium bromide (CTAB). The solubilized protein was purified on a chromatographic matrix with immobilized nickel. A stepwise gradient of imidazole concentration was applied, and the recombinant protein was eluted with a buffer containing 250 mM imidazole. The expression, solubilization and purification protocol used led to the recovery of Trx-CpOsm with a yield of 17.8 mg/L of culture. The purified protein fraction was concentrated using Vivaspin™ 20 with a 10 kDa MWCO (GE Healthcare) and employed in antifungal assays against Candida strains. Agar diffusion tests with Candida Krusei ATCC 6258, Candida albicans ATCC 10231, Candida parapsilosis ATCC 90018, Candida parapsilosis ATCC 22019, Candida tropicalis ATCC 13803 and Candida tropicalis ATCC 750 revealed the antifungal activity of the protein, establishing the formation of growth inhibition halos with diameters ranging from 21 mm to 35 mm. The antimicrobial activity of the protein solubilizing agent was also investigated. From the broth dilution test, cell growth inhibition activity promoted by Trx-CpOsm and CTAB were compared. The results indicate that Trx-CpOsm and CTAB have important antimycotic activity, with the persistent inhibitory action of the protein being notable even at lower concentrations, while CTAB loses its effectiveness at concentrations equal to or lower than 1.95 µg/mL. The residual persistence of CTAB after solubilization of inclusion bodies and recovery of the bioactive protein represents a possible reinforcement of the antifungal activity already demonstrated by the protein. These results open promising perspectives for the development of more effective antimycotic formulations, highlighting the biotechnological potential of this osmotin from Calotropis procera.