Optimization and scale-up production of Zika virus ΔNS1 in Escherichia coli: application of Response Surface Methodology

Abstract

Diagnosing Zika virus (ZIKV) infections has been challenging due to the cross-reactivity of induced antibodies with other flavivirus. The concomitant occurrence of ZIKV and Dengue virus (DENV) in endemic regions requires diagnostic tools with the ability to distinguish these two viral infections. Recent studies demonstrated that immunoassays using the C-terminal fragment of ZIKV NS1 antigen (ΔNS1) can be used to discriminate ZIKV from DENV infections. In order to be used in serological tests, the expression/solubility of ΔNS1 and growth of recombinant E. coli strain were optimized by Response Surface Methodology. Temperature, time and IPTG concentration were evaluated. According to the model, the best condition determined in small scale cultures was 21 °C for 20 h with 0.7 mM of IPTG, which predicted 7.5 g/L of biomass and 962 mg/L of ΔNS1. These conditions were validated and used in a 6-L batch in the bioreactor, which produced 6.4 g/L of biomass and 500 mg/L of ΔNS1 in 12 h of induction. The serological ELISA test performed with purified ΔNS1 showed low cross-reactivity with antibodies from DENV-infected human subjects. Denaturation of ΔNS1 decreased the detection of anti-ZIKV antibodies, thus indicating the contribution of conformational epitopes and confirming the importance of properly folded ΔNS1 for the specificity of the serological analyses. Obtaining high yields of soluble ΔNS1 supports the viability of an effective serologic diagnostic test capable of differentiating ZIKV from other flavivirus infections.

Keywords: E. coli; Heterologous protein production; Response Surface Methodology; Serological diagnosis; Soluble expression; Zika NS1.

Fig. 1

Response surface plots for biomass and production of soluble ΔNS1 as a function of time, temperature of induction and IPTG concentration. The model generated by the CCD for a–c biomass concentration measured as dry cell weight (DCW g/L) and d–f production of ΔNS1 the soluble fraction of cell extracts (mg/L). The temperature range was 16–30 °C, time between 12 and 24 h of induction and IPTG concentration between 0.4 and 1.0 mM. Color-coding indicates high (red) and low (blue) responses

Fig. 2

Predicted vs actual plots for biomass and ΔNS1. Linear regression plot for the predicted and actual responses for a biomass formation (g/L DCW) and b ΔNS1 (mg/L) obtained in soluble fraction of cell extracts

Fig. 3

Solubility of ΔNS1 during the induction in the bioreactor. After inoculation to achieve OD 0.1, culture was maintained at 37 °C until OD ~ 2.0 (pre-induction). Temperature was shifted to 21 °C and protein production induced with 0.7 mM IPTG. To determine solubility and biomass concentration, aliquots were taken at regular intervals after inoculation. After lysis, soluble and insoluble protein extracts were separated by SDS-PAGE and stained by Coomassie Blue. NI = non-induced. OD converted to biomass (g/L) according to the relation 1.0 OD = 0.34 g/L

Fig. 4

Antigenicity and specificity of the recombinant protein ΔNS1. a Coomassie Blue staining (left panels) and Western blots (right panels) obtained with 1 µg of purified ΔNS1 or full length Brazilian ZIKV NS1 and DENV2 NS1 (strain NGC). Western blots were probed with a mAb anti-His-Tag (ZIKV proteins) or anti-DENV2-NS1. b Reaction of human immune sera from eight DENV-infected subjects (serotypes 1 to 4) with DENV NS1, ZIKV NS1 and ΔNS1 in ELISA. c Reaction of ZIKV⁺ DENV⁺ human serum sample with intact and heat denatured DENV NS1, ZIKV NS1 and ΔNS1. Statistical significance was assessed by two-way ANOVA and the Bonferroni test