Ciencias Exactas y Ciencias de la Salud

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Heterologous expression, purification, and functional assessment of crotamine in E. coli using fusion tag strategies and cell-based assays
(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2025-06-13) Arjona Galiano, Daniel Alejandro; Benavides Lozano, Jorge; emimmayorquin; Meléndez Martínez, David; Antunes Ricardo, Marilena; School of Engineering and Sciences; Campus Monterrey; Vargas Cortez, Teresa
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.
Recombinant expression and in silico evaluation of AreV1, an Arenin variant.
(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2020-12-18) Teruel Barandiarán, Estefanía; Benavides Lozano, Jorge; tolmquevedo; Aguilar Jiménez, Oscar Alejandro; Chávez Santoscoy, Rocío Alejandra; School of Engineering and Sciences; Campus Monterrey; Hernández Pérez, Jesús
In the last decades, plants and animals described in Traditional Medicine practices have gained importance as sources of bioactive molecules since a great number of compounds have been isolated and characterized to develop new drugs. Although most of the current ethnopharmacological studies have focused on research bioactive compounds from plants, members of the anuran family Hylidae have also demonstrated to possess a plethora of molecules in their skin secretions with diverse capabilities. Among peptides, proteins, and low molecular weight compounds secreted by amphibians, protease inhibitors (PIs) have been proposed to be used as antimicrobial and antitumoral drugs that may possess high efficacy and efficiency. Recently, Arenin a Kunitz-like PI was isolated from the skin secretions of the canyon tree frog Dryophytes arenicolor. The DNA sequence encoding Arenin was modified by site-directed mutagenesis via PCR and the resultant variant, AreV1, was cloned, expressed, and purified by IMAC. Its 3D-structure was analyzed in silico through I-TASSER and COFACTOR servers and the structure modelling platform UCSF Chimera to assess the impact of the mutagenesis on the structure and further functionality of AreV1. Dissimilarities between AreV1 and Arenin were found at the C-terminus of the DNA sequence, altering ten amino acids (Leu45, Pro47, Trp48, Lys49, Ile50, Val51, Arg52, Pro53, Pro54 and Ala55). Despite these changes, according to the in silico analysis, the structural stability of AreV1 remained almost identical to Arenin, although the size of the variant polypeptide is three amino acids smaller. The analysis on AreV1 suggests that it has a great potential as trypsin regulator with a Lys13 as the residue located at the centre of the reactive site (P1 site). A tyrosine triplet (Tyr21, Tyr22 and Tyr23) associated with analgesic properties was also observed in AreV1.This research work aims to expand the knowledge on the activity optimization of naturally occurring molecules by presenting the recombinant production, purification and structural analysis of a variant from Arenin.

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