Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia
Abstract Background Although E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-...
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BMC
2024-01-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-024-02297-1 |
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| author | Heba Shawky Ashraf A. Tabll Reem M. Elshenawy Naiera M. Helmy Rehab I. Moustafa Yasser K. Elesnawy Marwa M. Abdelghany Yasmine S. El-Abd |
| author_facet | Heba Shawky Ashraf A. Tabll Reem M. Elshenawy Naiera M. Helmy Rehab I. Moustafa Yasser K. Elesnawy Marwa M. Abdelghany Yasmine S. El-Abd |
| author_sort | Heba Shawky |
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| description | Abstract Background Although E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection. Methods The core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab−) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab+) samples that included viremic (chronic/relapsers) and recovered patients’ sera. The color intensity (OD450) and S/Co values were estimated. Results The integration of 0.1–0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab−sera. The ideal S/Co threshold predicting active viremia (> 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection. Conclusion The applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings. |
| format | Article |
| id | doaj-art-27e208de221445c593edcbf1faf3dd24 |
| institution | Kabale University |
| issn | 1475-2859 |
| language | English |
| publishDate | 2024-01-01 |
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| series | Microbial Cell Factories |
| spelling | doaj-art-27e208de221445c593edcbf1faf3dd242024-12-29T12:53:25ZengBMCMicrobial Cell Factories1475-28592024-01-0123111910.1186/s12934-024-02297-1Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremiaHeba Shawky0Ashraf A. Tabll1Reem M. Elshenawy2Naiera M. Helmy3Rehab I. Moustafa4Yasser K. Elesnawy5Marwa M. Abdelghany6Yasmine S. El-Abd7Therapeutic Chemistry Department, Pharmaceutical Industries and Drug Research Institute, National Research CentreMicrobial Biotechnology Department, Biotechnology Research Institute, National Research CentreMicrobial Biotechnology Department, Biotechnology Research Institute, National Research CentreMicrobial Biotechnology Department, Biotechnology Research Institute, National Research CentreMicrobial Biotechnology Department, Biotechnology Research Institute, National Research CentreNational Committee for Control of Viral Hepatitis (NCCVH), Ministry of Health and PopulationAhmed Maher Teaching HospitalMicrobial Biotechnology Department, Biotechnology Research Institute, National Research CentreAbstract Background Although E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection. Methods The core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab−) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab+) samples that included viremic (chronic/relapsers) and recovered patients’ sera. The color intensity (OD450) and S/Co values were estimated. Results The integration of 0.1–0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab−sera. The ideal S/Co threshold predicting active viremia (> 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection. Conclusion The applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings.https://doi.org/10.1186/s12934-024-02297-1GlycylglycineE. coli (BL21)ChaperoneHCVEnzyme immunoassayCore |
| spellingShingle | Heba Shawky Ashraf A. Tabll Reem M. Elshenawy Naiera M. Helmy Rehab I. Moustafa Yasser K. Elesnawy Marwa M. Abdelghany Yasmine S. El-Abd Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia Microbial Cell Factories Glycylglycine E. coli (BL21) Chaperone HCV Enzyme immunoassay Core |
| title | Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia |
| title_full | Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia |
| title_fullStr | Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia |
| title_full_unstemmed | Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia |
| title_short | Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia |
| title_sort | glycylglycine promotes the solubility and antigenic utility of recombinant hcv structural proteins in a point of care immunoassay for detection of active viremia |
| topic | Glycylglycine E. coli (BL21) Chaperone HCV Enzyme immunoassay Core |
| url | https://doi.org/10.1186/s12934-024-02297-1 |
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