Ciencias Exactas y Ciencias de la Salud
Permanent URI for this collectionhttps://hdl.handle.net/11285/551039
Pertenecen a esta colección Tesis y Trabajos de grado de las Maestrías correspondientes a las Escuelas de Ingeniería y Ciencias así como a Medicina 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, TeresaCrotamine 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.
- Ultrasound-assisted extraction phenolic compounds from mexican raspberries (Rubus idaeus): cytotoxicity, antioxidant and anti- inflammatory activity(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022-07-06) López Corona, Alejandra Vanessa; Garcia Varela, Rebeca; puemcuervo, emimmayorquin; Sánchez López, Angélica Lizeth; García García, Jorge Donato; School of Engineering and Sciences; Campus Monterrey; Garcia Amezquita, Luis EduardoUltrasound-assisted extraction is a technology used as an alternative to conventional extraction methods to increase yield. In this research, the nutritional compositions, antioxidative, anti-inflammatory, and cytotoxic activity of organic Mexican raspberry were investigated to evaluate the effectiveness of ultrasound-assisted extraction of phenolic compounds from raspberry. Proximate composition was determined as 84.79% in moisture, 8.7 g/100 g d.b. protein, 5.9 g/100 g d.b. in fat, 40.6 g/100 g d.b. in total dietary fiber, and 3.4 g/100 g d.b. in ash. Ultrasound-assisted extraction was used to increase raspberry phenolic compounds extraction yield. Therefore, two samples were analyzed, raspberry extracts pretreated with ultrasound-assisted extraction (UAEp) and conventional raspberry extraction (N-UAEp). Total phenolics content was higher in UAEp (10.7 GAE mg/g DM) than in N-UAEp (8.2 GAE mg/g DM). Total anthocyanins content was similar in both UAEp and N-UAEp, 7.2 C3GE mg/g DM and 7.4 C3GE mg/g DM, respectively. Antioxidant activity measured by ABTS and DPPH assays in UAEp and N- UAEp extracts were 14.4 AAE mg/g DM and 8.3 AAE mg/g DM, 8.2 AAE mg/g DM and 6.1 AAE mg/g DM, respectively. UAEp raspberry extract showed a significant difference in antioxidative activity compared to N-UAEp. Ultra-high performance liquid chromatography analysis was performed for both sample groups, three anthocyanins were identified and quantified as most prevalent: cyanidin 3-glucoside, cyanidin 3- sophoroside and pelargonidin 3-glucoside. Arizona methodology was performed for the purpose of fractioning phenolic compound content found in raspberries. NIH/3T3 and CaCo-2 cells were used to determine cytotoxic capacity, here 70 treatments were assessed on each cell line. Treatments were design according to sample treatment (UAEp and N-UAEp), fractions (Arizona methodology) and doses (400-0 μg/L). The majority of UAEp treatments displayed high viability in NIH/3T3 and low viability for CaCo- 2, compared to N-UAEp. Based on cytotoxicity results, 16 treatments were chosen to test antioxidant and anti-inflammatory activity. Antioxidant activity was determined by the Oxygen Radical Antioxidant Capacity assay (27.4 – 72.2 % Cellular Antioxidant Activity) in CaCo-2 cells. RAW 264.7 cells were induced inflammation by lipopolysaccharides exposure to assess anti-inflammatory potential of all 16 treatments (38.9 – 10.9 % nitric oxide inhibition). Most cytotoxic, antioxidant and anti-inflammatory results display a dose- dependent behavior. Ultrasound-assisted extraction showed a significant favorable effect for the optimal extraction of phenolic compounds from raspberries.