Expresión y purificación de la enzima dihidrofolato reductasa de tipo salvaje y mutantes de Plasmodium vivax

• Autor: Perez Rodriguez, Victoriano Raul
• Formato: tesis de grado
• Fecha de Publicación: 2025
• Institución: Universidad Nacional De La Amazonía Peruana
• Repositorio: UNAPIquitos-Institucional
• Lenguaje: español
• OAI Identifier: oai:repositorio.unapiquitos.edu.pe:20.500.12737/12185
• Enlace del recurso: https://hdl.handle.net/20.500.12737/12185
• Nivel de acceso: acceso abierto
• Materia: Expresión génica; Síntesis de proteínas; Enzimas; Dihidrofolato reductasa; Malaria; Plasmodium vivax; Biotecnología; https://purl.org/pe-repo/ocde/ford#1.06.03

Due to its essential role in nucleotide synthesis, dihydrofolate reductase (DHFR) is a crucial enzyme present in various organisms, ranging from bacteria to mammals, including humans. In malaria-causing parasites, DHFR plays a vital role in parasite survival, contributing to its ability to resist and evade antifolate treatments. Although extensive genetic and enzymatic information is available for Plasmodium falciparum DHFR, data on Plasmodium vivax remain limited. Therefore, the main objective of this study was to standardize a protocol for the expression and purification of wild-type and mutant variants of P. vivax DHFR. Expression vectors carrying the recombinant genes were cloned and expressed in Escherichia coli Rosetta. After enzyme induction, they were purified using standard chromatographic techniques and characterized using spectrophotometric and electrophoretic methods, confirming their monomeric nature (~29 kDa). Affinity chromatography yielded concentrations of 9.93, 8.12, and 10.32 mg/ml, while size-exclusion chromatography produced 8, 7, and 9 mg/ml, resulting in total production of 48, 42, and 54 mg per liter of culture for each variant, respectively. The results demonstrate that the enzymes were successfully expressed and purified, obtaining sufficient quantities of highly pure enzyme. This achievement represents a significant advancement for future structural and functional studies of the enzyme.