Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in<i>Escherichia coli</i>

Liu, Jing; Zhu, Lizhi; Kong, Yu‐Ying; Li, Guangdi; Wang, Yuan

doi:10.3748/wjg.v11.i4.503

Cited by 5 publications

(5 citation statements)

References 24 publications

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“…Expressed in the absence of E2, E1 aggregates and does not fold correctly, making structural analysis impossible [26, 27]. This is consistent with reports that E1 might contain the hydrophobic peptide required for envelope fusion [28, 29].…”

Section: Structure Of E1 and E2supporting

confidence: 73%

Structural and Antigenic Definition of Hepatitis C Virus E2 Glycoprotein Epitopes Targeted by Monoclonal Antibodies

Sautto

Tarr

Mancini

et al. 2013

Clinical and Developmental Immunology

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Hepatitis C virus (HCV) is the major cause of chronic liver disease as well as the major indication for liver transplantation worldwide. Current standard of care is not completely effective, not administrable in grafted patients, and burdened by several side effects. This incomplete effectiveness is mainly due to the high propensity of the virus to continually mutate under the selective pressure exerted by the host immune response as well as currently administered antiviral drugs. The E2 envelope surface glycoprotein of HCV (HCV/E2) is the main target of the host humoral immune response and for this reason one of the major variable viral proteins. However, broadly cross-neutralizing monoclonal antibodies (mAbs) directed against HCV/E2 represent a promising tool for the study of virus-host interplay as well as for the development of effective prophylactic and therapeutic approaches. In the last few years many anti-HCV/E2 mAbs have been evaluated in preclinical and clinical trials as possible candidate antivirals, particularly for administration in pre- and post-transplant settings. In this review we summarize the antigenic and structural characteristics of HCV/E2 determined through the use of anti-HCV/E2 mAbs, which, given the absence of a crystal structure of this glycoprotein, represent currently the best tool available.

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Section: Structure Of E1 and E2supporting

confidence: 73%

Structural and Antigenic Definition of Hepatitis C Virus E2 Glycoprotein Epitopes Targeted by Monoclonal Antibodies

Sautto

Tarr

Mancini

et al. 2013

Clinical and Developmental Immunology

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“…Determining the structure of the E1E2 heterodimer has proven problematic. E2 is required for the correct folding of E1, so that E1’s structure is still uncertain ( 35 , 36 ). It is thought that the glycans on both the heavily glycosylated E1 and E2 are involved in folding of the E1E2 heterodimer ( 37 ).…”

Section: Nab Epitopesmentioning

confidence: 99%

The Humoral Immune Response to HCV: Understanding is Key to Vaccine Development

2014

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Hepatitis C virus (HCV) remains a global problem, despite advances in treatment. The low cost and high benefit of vaccines have made them the backbone of modern public health strategies, and the fight against HCV will not be won without an effective vaccine. Achievement of this goal will benefit from a robust understanding of virus–host interactions and protective immunity in HCV infection. In this review, we summarize recent findings on HCV-specific antibody responses associated with chronic and spontaneously resolving human infection. In addition, we discuss specific epitopes within HCV’s envelope glycoproteins that are targeted by neutralizing antibodies. Understanding what prompts or prevents a successful immune response leading to viral clearance or persistence is essential to designing a successful vaccine.

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“…Having discussed the cloning success, it is notable that the number of positive E1/E2 transformants was limited compared to the core. This observation could signify a basal expression of the E1/E2 protein reported with hydrophobicity-associated toxicity [ 27 ], despite using a moderate promoter (T5)-based expression vector [ 28 ]. Accordingly, several conditions were tailored in glycylglycine-free cultures to overcome this toxicity and obtain the maximum yield of the recombinant E1/E2 protein, including the tight control of basal expression by adding 1% (v/v) glucose [ 20 ], low pre/post-induction incubation temperature (30 and 16℃, respectively), and low IPTG concentration (0.2 mM).…”

Section: Discussionmentioning

confidence: 99%

“…While the exact mechanism of glycylglycine in this context is unclear, it could be proposed that high concentrations of the dimer in the growth culture might generate an osmophobic effect, which would induce the overexpression of the heat shock chaperones like DnaK and GroEL(S) in E. coli that subsequently promote a ubiquitin–proteasome pathway and/or autophagy-mediated degradation of cytosol-accumulated proteins [ 29 – 31 ]. By the same logic, the preferential promotive effect of glycylglycine observed on the cell density and protein yield in the recombinant E1/E2 cultures, despite the reported protein toxicity [ 27 ], could be related to the preferential recognition/binding of DnaK with hydrophobic motifs of five residues [ 32 ] that are typically abundant in the E1/E2 transmembrane (TM) domains [ 33 , 34 ]. This hypothesis also justifies the divergence between our results and those of Rani et al .…”

Section: Discussionmentioning

confidence: 99%

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

Shawky,

Tabll,

Elshenawy

et al. 2024

Microb Cell Fact

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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.

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Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed inEscherichia coli

Abstract: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.

Cited by 5 publications

References 24 publications

Structural and Antigenic Definition of Hepatitis C Virus E2 Glycoprotein Epitopes Targeted by Monoclonal Antibodies

Structural and Antigenic Definition of Hepatitis C Virus E2 Glycoprotein Epitopes Targeted by Monoclonal Antibodies

The Humoral Immune Response to HCV: Understanding is Key to Vaccine Development

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

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