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

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.

Fig 1. Incubation at HHP promotes solubilization of NS1-IBs.

Suspensions of NS1-IBs were subjected to 2.4 kbar for 90 min and to 0.4 kbar for 14 h30 min (2.4/0.4 kbar) or to 1 bar for 16 h. A, LS vs pH; B, LS vs GdnHCl concentration at pH 8.5. The LS value, obtained for the suspension at a pH 7.0 that was not subjected to HHP and read immediately after dilution, was considered to be 100%. C, LS vs pH and presence of Arg. The LS value obtained for the suspension at pH 7.0, determined after 16 h incubation, was considered to be 100%. 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. Values are expressed as LS mean ± SD. Each condition was analysed in quadruplicate. The results shown are representative of 3 independent assays.

Fig 2. NS1 is found in the soluble fraction of the samples subjected to HHP at alkaline pH.

A, SDS-PAGE analysis of the supernatant of the suspension incubated at 1 bar at different pH; B, SDS-PAGE analysis of the supernatant of the suspension incubated at HHP (2.4 kbar for 90 min and 0.4 kbar for 14h 30 min) at different pH. The results shown are representative of 3 independent assays.

Fig 3. Application of HHP at pH 11.0–12.0 dissociate NS1-IBs oligomers.

SEC of supernatants of DENV 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. The results shown are representative of 3 independent assays.

Fig 4. HHP in association with alkaline pH and the presence of Arg induce only partial NS1 unfolding.

Suspensions of NS1-IB were subjected to 2.4 kbar/0.4 kbar or to 1 bar treatment. A, λ maximum vs pH; B, λ maximum vs GdnHCl concentration. An excitation at 290 nm was used for intrinsic fluorescence determination and the emission was measured between 300 and 400 nm. Each condition was analysed at least in triplicate. Values are expressed as mean ± SD. The results shown are representative of 3 independent assays.

Fig 5. NS1 solubilized by application of HHP at pH of 10.0–11.5 and dialyzed at a pH of 8.5 forms mostly trimers, dimers and monomers.

DENV NS1-IB suspensions were subjected to 2.4 kbar/0.4 kbar 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. The results shown are the representative of 2 independent assays.

Fig 6. Concentration of NS1 in the supernatants of NS1-IB subjected to HHP.

Suspensions of NS1-IB were subjected to 2.4 kbar/0.4 kbar or to 1 bar. A) NS1 concentration vs pH; B) NS1 concentration vs GdnHCl concentration. Values are expressed as LS mean ± SD. Each condition was analysed at least in triplicate and the assay was performed 4 times. C) NS1 refolded at HHP and the pH indicated; D, NS1 refolded at HHP. Column 1, pH 10.5 + 0.4 M arg, column 2, pH 11.0 + 0.4 M Arg, column 3, pH 11.0 + 0.4 M Arg + 1 mM GSH + 0.1 mM GSSG. IB, NS1-IB suspension. The results shown are representative of 6 independent assays.

Fig 7. DENV NS1 refolded at HHP is antigenic.

A) Titers of NS1-specific IgG measured by ELISA using NS1 refolded with HHP at pH 10.5 in different conditions. The ELISA was employing with a control sera obtained from patients that had been previously infected with DENV (gray bars) or not (white bars). B) Evaluation of the preservation of conformational epitopes in NS1. NS1 obtained at HHP, pH 10.5 + Arg + GSH/GSSG was previously denatured (100 °C, 10 min) or not and analyzed by ELISA for reactivity with a serum from a patient previously infected with DENV. For A and B, the IB compression was performed in 2.4 kbar / 0.4 kbar. C) Titers of NS1 refolded at HHP at pH 10.5 + Arg + GSH/GSSG by incubation at 2.4 kbar (90 min.) and 0.4 kbar for the time indicated in the figure. Values are expressed as mean ± SD of the data. All NS1 samples had low reactivity with control serum in ELISA. Control NS1, obtained from the same clone used in this study and refolded using traditional protocols performed at atmospheric pressure. * p < 0.05, ** p <0.01, *** p<0.001 (Two way ANOVA with Bonferroni post-test). The results shown are the representative of 3 independent assays.