RESUMO
Abstract This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.
Assuntos
Humanos , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Pichia/genética , Proteínas Recombinantes/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Clonagem Molecular , Meios de Cultura/química , Fator de Crescimento Epidérmico/genética , Fermentação , Concentração de Íons de HidrogênioRESUMO
This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.(AU)
Assuntos
Pichia , Fator de Crescimento Epidérmico , Rede Nervosa , Engenharia Genética , DNA RecombinanteRESUMO
This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.