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

Abstract

Background: Proteins 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.

Results: Pressure-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.

Conclusions: The 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.

Keywords: Alkaline pH; Dengue virus and Zika virus; High hydrostatic pressure; Inclusion bodies; NS1; Protein refolding.

Fig. 1

Incubation at high pressure promotes solubilization of NS1-IBs. Suspension of NS1-IBs was subjected to 2.4 kbar for 90 min and at 0.4 kbar for 14 h 30 min (2.4/0.4 kbar) or at 1 bar for 16 h. a Curve of LS vs GndHCl; b curve of LS vs pH; c SDS-PAGE analysis of the supernatant of the suspension subjected to 1 bar; d SDS-PAGE analysis of the supernatant of the suspension maintained at 2.4 kbar/0.4 kbar and e curve of LS vs Arg (pH 10.5). The LS measurements were carried out in a spectrofluorimeter with an excitation of 320 nm; the emission was determined between 315 and 325 nm, and the areas of the peaks were used for plotting. The LS value obtained for the suspension at a pH of 8.0 read immediately at 1 bar was considered to be 100%. For SDS-PAGE samples were boiled and reduced. The results are representative of experiments performed three times

Fig. 2

High pressure in association with alkaline pH and the presence of Arg induce only partial unfolding of NS1-IB. Suspensions of NS1-IB were subjected at 2.4 kbar/0.4 kbar or at 1 bar. a Intrinsic (Trp) fluorescence of NS1-IB; b λ maximum of fluorescence vs concentration of GdnHCl; c λ maximum of fluorescence vs pH and d λ maximum of fluorescence vs concentration of Arg. An excitation at 290 nm was used for intrinsic fluorescence determination and the emission was measured between 300 and 400 nm. The results are representative of experiments performed three times

Fig. 3

Application of HHP at alkaline pH solubilizes NS1-IBs and the presence of Arg helps to dissociate oligomers. SEC from supernatants of ZIKV NS1-IB suspensions subjected to 2.4 kbar/0.4 kbar. A volume of 500 μL of the supernatants of the suspensions subjected to HHP was applied to a Superdex 200 10/300 column (GE Biosciences). The elution buffer was 50 mM CAPS at a pH of 11.0

Fig. 4

Higher concentrations of NS1 are found in the supernatant of the suspensions subjected to HHP at alkaline pH. Concentration of NS1 in ZIKV NS1-IB supernatants submitted to 2.4 kbar / 0.4 kbar or to 1 bar and dialyzed against 50 mM TrisHCl at a pH of 8.5. a concentration of NS1 vs concentration of GdnHCl; b concentration of NS1 vs pH; c concentration of NS1 vs concentration of Arg. The results are representative of experiments performed three times

Fig. 5

NS1 solubilized by application of HHP at a pH of 11.5 forms oligomers after dialysis at a pH of 8.5. ZIKV NS1-IB suspensions were subjected to 2.4 kbar/0.4 kbar at a pH of 11.5 and dialyzed against 50 mM Tris HCl at a pH of 8.5. A volume of 500 μL of the supernatant of the suspensions was applied to a Superdex 200 10/300 column (GE Biosciences). The elution buffer was TrisHCl 50 mM at a pH of 8.5

Fig. 6

Conformational analysis of refolded NS1. a Supernatants from suspensions of NS1-IBs refolded by the application of HHP (2.4 kbar / 0.4 kbar) were dialyzed and analyzed by SDS-PAGE. Condition 1: pH 11.0 + DTT (2 mM); condition 2: pH 11.0 + Arg (0.4 M), condition 3: pH 11.0 + Arg + GSH (1 mM) + GSSG (0.1 mM); condition 4: pH 11.5 + Arg; condition 5: pH 11.5 + Arg + GSH/GSSG. Sample IB: The suspension was homogenized for application in the SDS-PAGE. All the samples were heated at 95 °C in the presence of DTT (100 mM) before application in SDS-PAGE; b Far-UV CD spectra of NS1 (2 μM recorded in 0.2 cm path length quartz cell) refolded at pH 11 in the presence of Arg

Fig. 7

NS1 of ZIKV refolded at HHP and pH of 11.0 is antigenic. a NS1 was used as a solid phase antigen in ELISA assays employing control sera obtained from patients that had been previously infected with ZIKV (black bars) or not (white bars). Values are expressed as mean ± error of antigen-specific IgG antibody titers; b Evaluation of the preservation of conformational epitopes of the ZIKV NS1. NS1 obtained at HHP and pH 11.0 was submitted to denaturation (heating at 100 °C for 10 min followed by heat shock at 0 °C) or not and analyzed by ELISA for reactivity with a serum from a patient previously infected with ZIKV. The values obtained are expressed as mean ± error of the absorbance obtained in the assay. * p < 0.05; ** p < 0.01; *** p < 0.001 (Two-way ANOVA with Bonferroni test). The IB compression was performed in 2.4 kbar / 0.4 kbar. Control: NS1 obtained from the same clone used in this study and refolded using traditional protocol at atmospheric pressure