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

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 recombi...

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Bibliographic Details
Published in:PloS one 2019-01, Vol.14 (1), p.e0211162-e0211162
Main Authors: Chura-Chambi, Rosa Maria, da Silva, Cleide Mara Rosa, Pereira, Lennon Ramos, Bartolini, Paolo, Ferreira, Luis Carlos de Souza, Morganti, Ligia
Format: Article
Language:English
Subjects:
Alkalies
Antigens
Arginine
Biology and Life Sciences
Compression
Dengue
Dengue fever
Dengue Virus - chemistry
Dengue Virus - genetics
Dimers
E coli
Escherichia coli
Gene expression
Growth hormones
Hydrogen-Ion Concentration
Hydrostatic Pressure
Inclusion bodies
Medicine and Health Sciences
NS1 protein
pH effects
Physical Sciences
Pressure
Protein folding
Protein Refolding
Proteins
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Research and Analysis Methods
Solubilization
Vaccines
Vector-borne diseases
Viral diseases
Viral Nonstructural Proteins - chemistry
Viral Nonstructural Proteins - genetics
Viruses
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0211162