The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

Autor(es)

Malavasi, N. V. ^[1] ; Cordeiro, Y. ^[2] ; Rodrigues, D. ^[1] ; Chura-Chambi, R. M. ^[1] ; Lemke, L. S. ^[1] ; Morganti, L. ^[1]

Afiliação do(s) autor(es)

^[1] CNENSP, Ctr Biotecnol, IPEN, Sao Paulo - Brazil

^[2] Univ Fed Rio de Janeiro, Fac Farm, Rio De Janeiro - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Process Biochemistry; v. 49, n. 1, p. 54-60, JAN 2014.
• Citações Web of Science: 0

Resumo

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 degrees C are more efficiently dissociated at 2.4 kbar than those produced at 47 degrees C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (-9 degrees C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 C to 50 degrees C is proportional to the temperature, and a higher yield was obtained at 20 C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 C), of their dissociation at HHP (-9 degrees C) and of eGFP refolding (20 degrees C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology. (C) 2013 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 08/57338-5 - Estudos de renaturacao de proteinas agregadas utilizando altas pressoes hidrostaticas
• Beneficiário: Natália Malavasi Vallejo
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 10/13353-0 - Caracterização de estados intermediários do enovelamento e obtenção de proteínas biofuncionais pela utilização de altas pressões hidrostáticas
• Beneficiário: Ligia Ely Morganti Ferreira Dias
• Modalidade de apoio: Auxílio à Pesquisa - Regular