Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia.

Autor: Shawky H; Therapeutic Chemistry Department, Pharmaceutical Industries and Drug Research Institute, National Research Centre, Dokki, Cairo, 12622, Egypt., Tabll AA; Microbial Biotechnology Department, Biotechnology Research Institute, National Research Centre, Dokki, Cairo, 12622, Egypt., Elshenawy RM; Microbial Biotechnology Department, Biotechnology Research Institute, National Research Centre, Dokki, Cairo, 12622, Egypt., Helmy NM; Microbial Biotechnology Department, Biotechnology Research Institute, National Research Centre, Dokki, Cairo, 12622, Egypt., Moustafa RI; Microbial Biotechnology Department, Biotechnology Research Institute, National Research Centre, Dokki, Cairo, 12622, Egypt., Elesnawy YK; National Committee for Control of Viral Hepatitis (NCCVH), Ministry of Health and Population, Cairo, Egypt., Abdelghany MM; Ahmed Maher Teaching Hospital, Cairo, Egypt., El-Abd YS; Microbial Biotechnology Department, Biotechnology Research Institute, National Research Centre, Dokki, Cairo, 12622, Egypt. ys.el-abd@nrc.sci.eg.
Jazyk: angličtina
Zdroj: Microbial cell factories [Microb Cell Fact] 2024 Jan 18; Vol. 23 (1), pp. 25. Date of Electronic Publication: 2024 Jan 18.
DOI: 10.1186/s12934-024-02297-1
Abstrakt: 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 (OD4 50 ) 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.
(© 2024. The Author(s).)
Databáze: MEDLINE
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