Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from <i>E. coli</i> system

Gao, Xiangzheng; Shan-shan, Peng; Mei, Shengsheng; Liang, Keying; Khan, Muhammad Saleem Iqbal; Vong, Eu Gene; Zhan, Jun

doi:10.1080/10826068.2021.1941106

Cited by 16 publications

(11 citation statements)

References 19 publications

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“…So far, we report for the first time the successful expression of SARS-CoV-2 rRBD in four different E. coli expression strains: E. coli BL21(DE3) pLysS, E. coli BL21(DE3) Arctic RIL, E. coli BL21(DE3) C43 and E. coli BL21(DE3) Rosetta Gami. Our results corroborate with Gao and colleagues work of successful expression of RBD with a C-terminal histidine tag in E. coli BL21 (DE3) Rosetta Gami from pET-28a(+) using 1 mM IPTG as an inducer [11]. The same study reported that the renatured RBD could efficiently bind to ACE2 as was also shown by He and colleagues for RBD expressed from E. coli (BL21) [10].…”

Section: Coronavirus Disease 2019 (Covid-19supporting

confidence: 91%

“…Albeit its usefulness and numerous advantages, mammalian expression is time consuming, expensive, and requires sophisticated laboratory settings. Only a few studies reported RBD expression in prokaryotic expression systems for investigating its structural and binding characteristics [10,11]. Less than a handful of studies reported the reactivity of RBD produced in E. coli with human sera of infected SARS-CoV-2 [12].…”

Section: Introductionmentioning

confidence: 99%

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Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA

Hussein

Ali

El-Hakim

et al. 2022

HAB

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BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. OBJECTIVE: The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. METHODS: We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in five different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. RESULTS: The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88% specificity, 78.5% agreement, a positive predictive value (PPV) of 92.3%, and a negative predictive value of 56.5%. Moreover, the optical density (OD) values of positive samples significantly correlated with the commercial kit titers. CONCLUSIONS: In conclusion, specific human antibodies against SARS-CoV-2 spike protein were detected by rapid in-house ELISA in sera of human COVID-19-infected patients. The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications, particularly in developing countries. Future studies are planned for the use of the generated SARS-CoV-2 rRBD-6×His protein in vaccine development and other diagnostic applications.

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Section: Coronavirus Disease 2019 (Covid-19supporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA

Hussein

Ali

El-Hakim

et al. 2022

HAB

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“…In particular, the Receptor-Binding-Domain (RBD) of spike protein was overexpressed using E. coli BL21(DE3) or its derivative Rosetta(DE3). The target protein was then recovered from inclusion bodies and subsequently purified (Fitzgerald et al 2021 ; Gao et al 2021 ). However, it has been reported that the RBD of spike protein features poor immunogenicity (Tan et al 2021 ), most probably because of its low molecular mass.…”

Section: Introductionmentioning

confidence: 99%

High-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2

et al. 2022

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Objectives The aim of the present work was to identify a time-saving, effective, and low-cost strategy to produce in Escherichia coli a protein chimera representing a fusion anti-SARS-CoV-2 candidate vaccine, consisting of immunogenic and antigenic moieties. Results We overexpressed in E. coli BL21(DE3) a synthetic gene coding for CRM197-RBD, and the target protein was detected in inclusion bodies. CRM197-RBD was solubilized with 1 % (w/v) of the anionic detergent N-lauroylsarcosine (sarkosyl), the removal of which from the protein solution was conveniently accomplished with Amberlite XAD-4. The detergent-free CRM197-RBD was then separated from contaminating DNA using polyethylenimine (PEI), and finally purified from PEI by salting out with ammonium sulfate. Structural (CD spectrum) and functional (DNase activity) assays revealed that the CRM197-RBD chimera featured a native and active conformation. Remarkably, we determined a yield of purified CRM197-RBD equal to 23 mg per litre of culture. Conclusions To produce CRM197-RBD, we devised the use of sarkosyl as an alternative to urea to solubilize the target protein from E. coli inclusion bodies, and the easy removal of sarkosyl by means of Amberlite XAD-4.

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“…With the perspective of selecting anti-RBD adhirons for a new biosensor, we envisaged a Spike RBD production protocol more effective than the strategies already available. If bacterial expression provides deglycosylated and mainly insoluble RBD [ [15] , [16] , [17] , [18] ] ( Table 1 ), eukaryotic systems based on cells grown in both flasks and bioreactors were successful to express RBD [ 10 , [19] , [20] , [21] , [22] , [23] ] but had specific shortcomings ( Table 1 ). For instance, RBD production in yeast reached yields of 60 mg/L [ 22 ] but showed high level of immunogenic N-glycans which made it unsuitable for serological tests, whereas RBD expressed in insect cells was O-glycosylated [ 23 ] ( Table 1 ).…”

Section: Introductionmentioning

confidence: 99%

Expression, purification and characterization of SARS-CoV-2 spike RBD in ExpiCHO cells

March

Terdoslavich

Polez

et al. 2022

Protein Expression and Purification

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Reliable diagnosis is critical to identify infections of SARS-CoV-2 as well as to evaluate the immune response to virus and vaccines. Consequently, it becomes crucial the isolation of sensitive antibodies to use as immunocapture elements of diagnostic tools. The final bottleneck to achieve these results is the availability of enough antigen of good quality. We have established a robust pipeline for the production of recombinant, functional SARS-CoV-2 Spike receptor binding domain (RBD) at high yield and low cost in culture flasks. RBD was expressed in transiently transfected ExpiCHO cells at 32 °C and 5% CO 2 and purified up to 40 mg/L. The progressive protein accumulation in the culture medium was monitored with an immunobinding assay in order to identify the optimal collection time. Successively, a two-step chromatographic protocol enabled its selective purification in the monomeric state. RBD quality assessment was positively evaluated by SDS-PAGE, Western Blotting and Mass Spectrometry, while Bio-Layer Interferometry, flow cytometer and ELISA tests confirmed its functionality. This effective protocol for the RBD production in transient eukaryotic system can be immediately extended to the production of RBD mutants.

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Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from E. coli system

Cited by 16 publications

References 19 publications

Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA

Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA

High-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2

Expression, purification and characterization of SARS-CoV-2 spike RBD in ExpiCHO cells

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