Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus

Cleide Mara Rosa Da Silva,Rosa Maria Chura-Chambi,Yraima Cordeiro,Lennon Ramos Pereira,Ligia Morganti,Luís Carlos De Souza Ferreira

doi:10.1186/s12896-018-0486-2

Abstract

BackgroundProteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein.ResultsPressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods.ConclusionsThe present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.

Highlights

Proteins in inclusion bodies (IBs) present native-like secondary structures
Solubilization of non-structural 1 (NS1)-IB The first step in establishing an efficient protein refolding protocol is the efficient solubilization of protein aggregates
To determine whether the association of High hydrostatic pressure (HHP) and guanidine hydrochloride (GndHCl) promote efficient solubilization of zika virus (ZIKV) Inclusion bodies of NS1 (NS1-IB), suspensions were submitted to 2.4 kbar for 90 min and to 0.4 kbar for 14 h 30 min (2.4 kbar/0.4 kbar)

Summary

Introduction

Chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. High pH has been described as a mild technique to solubilize IBs with efficient subsequent refolding In this case the presence of a denaturing agent at low concentrations (2 M urea) is required for efficient refolding [13,14,15]. The concomitant use of physical and chemical treatments, i.e., high hydrostatic pressures and alkaline pH, is described in the present study for the solubilization and subsequent refolding of protein aggregates

Methods

Results

Conclusion