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Optimization and scale-up production of Zika virus ?NS1 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 (?NS1) can be used to discriminate ZIKV from DENV infections. In order to be used in serological tests, the expression/solubility of ?NS1 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 ?NS1. 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 ?NS1 in 12 h of induction. The serological ELISA test performed with purified ?NS1 showed low cross-reactivity with antibodies from DENV-infected human subjects. Denaturation of ?NS1 decreased the detection of anti-ZIKV antibodies, thus indicating the contribution of conformational epitopes and confirming the importance of properly folded ?NS1 for the specificity of the serological analyses. Obtaining high yields of soluble ?NS1 supports the viability of an effective serologic diagnostic test capable of differentiating ZIKV from other flavivirus infections.

SUBMITTER: Kanno AI

PROVIDER: S-EPMC6938527 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Kanno Alex Issamu AI Leite Luciana Cezar de Cerqueira LCC Pereira Lennon Ramos LR de Jesus Mônica Josiane Rodrigues MJR Andreata-Santos Robert R Alves Rúbens Prince Dos Santos RPDS Durigon Edison Luiz EL Ferreira Luís Carlos de Souza LCS Gonçalves Viviane Maimoni VM

AMB Express 20191231 1

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, th ...[more]

PMID: 31893321

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Project description:IntroductionA halophilic bacterium of the Halomonas elongata BK-AG25 has successfully produced ectoine with high productivity. To overcome the drawbacks of high levels of salt in the production process, a nonhalophilic bacteria of Escherichia coli (E. coli) was used to express the ectoine gene cluster of the halophilic bacteria, and the production of ectoine by the recombinant cell was optimized.MethodsThe ectoine gene cluster from the halophilic bacterium was isolated and inserted into an expression plasmid of pET30(a) and subsequently transformed into E. coli BL21 (DE3). Production of ectoine from the recombinant E. coli was investigated and then maximized by optimizing the level of nutrients in the medium, as well as the bioprocess conditions using response surface methodology. The experimental designs were performed using a central composite design.ResultsThe recombinant E. coli successfully expressed the ectoine gene cluster of Halomonas elongata BK-AG25 under the control of the T7 promoter. The recombinant cell was able to produce ectoine, of which most were excreted into the medium. The optimization of ectoine production with the response surface methodology showed that the level of salt in the medium, the incubation temperature, the optical density of the bacteria before induction, and the final concentration of the inducer gave a significant effect on ectoine production by the recombinant E. coli. Interestingly, the level of salt in the medium and the incubation temperature showed an inverse effect on the production of intracellular and extracellular ectoine by the recombinant cell. At the optimum conditions, the production yield was about 418 mg ectoine/g cdw (cell dry weight) after 12 hours of incubation.ConclusionThis study is the first report on the expression of an ectoine gene cluster of Halomonas elongata BK-AG25 in E. coli BL21, under the control of the T7 promoter. Optimization of the level of nutrients in the medium, as well as the bioprocess condition using response surface methodology, has successfully increased the production of ectoine by the recombinant bacteria.

Dataset Information

Optimization and scale-up production of Zika virus ?NS1 in Escherichia coli: application of Response Surface Methodology.

Publications

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

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