PLoS ONE (Jan 2019)

Protein refolding based on high hydrostatic pressure and alkaline pH: Application on a recombinant dengue virus NS1 protein.

  • Rosa Maria Chura-Chambi,
  • Cleide Mara Rosa da Silva,
  • Lennon Ramos Pereira,
  • Paolo Bartolini,
  • Luis Carlos de Souza Ferreira,
  • Ligia Morganti

DOI
https://doi.org/10.1371/journal.pone.0211162
Journal volume & issue
Vol. 14, no. 1
p. e0211162

Abstract

Read online

In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.