Examinando por Materia "Cromatografía de afinidad"

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    Producción de Factor de Crecimiento Epidermal Humano (hEGF) por métodos recombinantes
    (Universidad EAFIT, 2024) González Santos, Andrés Felipe; Corrales García, Ligia Luz
    Human Epidermal Growth Factor (hEGF) is a protein naturally produced by skin cells and essential in wound healing and tissue regeneration. In this study, the production of recombinant hEGF was carried out using Escherichia coli as the expression model. The hEGF gene was cloned into the pET32a vector and initially transformed into E. coli XL1-Blue for its amplification. Subsequently, the purified plasmid was used to transform E. coli BL21, a strain optimized for recombinant protein expression. hEGF expression was induced with 1 mM IPTG (Isopropyl ß-D-1-thiogalactopyranoside) at 16 °C for 16 hours, conditions that favored obtaining the protein in soluble form. Purification was performed through a two-stage process: nickel a inity chromatography (Ni-NTA) followed by anion exchange chromatography. The identity and purity of the protein were confirmed by performing acrylamide gel electrophoresis (SDS-PAGE) and Western blot based on the use of anti-His antibodies. This protocol allowed obtaining recombinant hEGF in its soluble and active form, establishing an e icient methodology for its production at a laboratory scale.