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

Rosa Maria Chura-Chambi,Paolo Bartolini,Ligia Morganti,Cleide Mara Rosa Da Silva,Luis Carlos De Souza Ferreira,Lennon Ramos Pereira,Malaya Kumar Sahoo

doi:10.1371/journal.pone.0211162

Abstract

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.

Highlights

Escherichia coli strains are the most usual alternative for production of heterologous proteins, for those that do not require post-translational modifications [1]
Our results demonstrated that association of high hydrostatic pressure (HHP) with alkaline conditions allowed the dissociation of aggregates into immunologically active soluble non-structural protein 1 (NS1) at high yields and, represents a novel alternative for the solubilization of recombinant proteins accumulated in inclusion bodies (IBs)
With the aim to solubilize IBs of a recombinant form of the dengue virus NS1 protein expressed in E.coli (NS1-IB) as mildly as possible, we used the combinations of HHP and alkaline pH as well as HHP and guanidine hydrochloride (GdnHCl)

Summary

Introduction

Escherichia coli strains are the most usual alternative for production of heterologous proteins, for those that do not require post-translational modifications [1]. Depending on the characteristics of the expressed protein, incubation temperature and expression levels, it may be produced in soluble form or as insoluble aggregates: the inclusion bodies (IBs) [2]. In IBs the proteins form amyloid-like structures in which molecules, with a conformation that include the native ones, are trapped [3]. Proteins in IBs frequently keep secondary and tertiary structures similar to those found in their native conformation [4] and may even show some degree of biological activity [5, 6]. The difficulties faced to obtain a fully active protein from IBs is a frequent drawback. The first step in the refolding processes is the solubilization of the insoluble

Methods

Results

Conclusion