Heterologous expression, purification, and functional assessment of crotamine in E. coli using fusion tag strategies and cell-based assays

Abstract

Crotamine is a peptide found in the venom of Crotalus durrisus terrificus, it has sparked interest in the scientific community because of its therapeutic potential, particularly because of its affinity to act on actively proliferating cells, acting as an anti-cancer agent. However, the research and possible applications of the peptide have been hindered by its availability, as it’s hard to get enough pure, and bioactive crotamine, as its natural source produces low quantities and recombinant methods have faced hurdles in having high yields and proper folding of the protein. This thesis aimed to develop and compare heterologous expression strategies to obtain crotamine using E. coli as the production vector, looking to obtain a scalable production method. The three plasmid designs were His6-MBP-Crotamine, His6-Crotamine, and Tagless-Crotamine. Each approach aimed to experiment on how the tags affect the solubility, yields, and purification process of the protein. Expression was done in E. coli BL21star, followed by cell lysis of the wet pellet, centrifugation to separate the soluble and insoluble fractions, Inmobilized Metal Affinity Chromatography (IMAC) to purify the peptide alongside its tags, TEV protease cleavage to remove the tags, and desalting of the product to lyophilize it into the final pure protein. Purity itself was confirmed through SDS-PAGE and yields quantified with BCA assay. Finally, the bioactivity of the purified crotamine was evaluated using MTS cell viability assays on Human colorectal adenocarcinoma (Caco-2) cells, human breast adenocarcinoma (MCF-7) cells and human primary dermal fibroblasts (HDF-a). The results showed that the His6-MBP tag significantly increased the solubility of crotamine, obtaining 83% of the protein in the soluble fraction. However, purification of His6-MBP-Crotamine after TEV cleavage using IMAC proved difficult due to crotamine’s intrinsic affinity for the affinity column, resulting in low purity of 11% and an overall low yield of 44.4%, with a final amount of 4.44 mg/L of production. Meanwhile, the Tagless-Crotamine yielded the highest amount of protein, obtaining 17.71 mg/L between the soluble and insoluble fractions, with the purified insoluble fraction obtaining a purity of 85%. The bioactivity assays with the purified crotamine showed cytotoxicity towards HDF-a and Caco-2 cells, with no significant effect on MCF-7 cells, suggesting potential differences in activity compared to the native crotamine. The Tagless-Crotamine proved to be the most promising strategy as it offered higher yields, an easier purification process, and a bioactive crotamine; However, its effects on non-tumoral cells warrant further investigation into its structure and post-translational modifications to compare it with that of the native peptide. Still, this work provides a scalable approach for recombinant crotamine production that facilitates further research of the protein into its diverse therapeutic applications.