Soluble overexpression, high-level production and purification of receptor binding domain of human VEGF8-109 in E. coli

Multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have spread around the world, but the neutralizing effects of antibodies induced by the existing vaccines have declined, which highlights the importance of developing vaccines against mutant virus strains. In this study, nine receptor-binding domain (RBD) proteins of the SARS-CoV-2 variants (B.1.1.7, B.1.351 and P.1 lineages) were constructed and fused with the Fc fragment of human IgG (RBD-Fc). These RBD-Fc proteins contained single or multiple amino acid substitutions at prevalent mutation points of spike protein, which enabled them to bind strongly to the polyclonal antibodies specific for wild-type RBD and to the recombinant human ACE2 protein. In the BALB/c, mice were immunized with the wild-type RBD-Fc protein first and boosted twice with the indicated mutant RBD-Fc proteins later. All mutant RBD-Fc proteins elicited high-level IgG antibodies and cross-neutralizing antibodies. The RBD-Fc proteins with multiple substitutions tended to induce higher antibody titers and neutralizing-antibody titers than the single-mutant RBD-Fc proteins. Meanwhile, both wild-type RBD-Fc protein and mutant RBD-Fc proteins induced significantly decreased neutralization capacity to the pseudovirus of B.1.351 and P.1 lineages than to the wild-type one. These data will facilitate the design and development of RBD-based subunit vaccines against SARS-COV-2 and its variants.

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Investigating Early Events in Receptor Binding and Translocation of Colicin E9 Using Synchronized Cell Killing and Proteolytic Cleavage

Journal of Bacteriology ◽

10.1128/jb.00047-08 ◽

2008 ◽

Vol 190 (12) ◽

pp. 4342-4350 ◽

Cited By ~ 14

Author(s):

Ying Zhang ◽

Mireille N. Vankemmelbeke ◽

Lisa E. Holland ◽

David C. Walker ◽

Richard James ◽

...

Keyword(s):

Cell Surface ◽

Receptor Binding ◽

Distal Region ◽

Cell Killing ◽

Binding Domain ◽

Receptor Binding Domain ◽

Cleavage Sites ◽

E Coli ◽

Translocation Domain ◽

Colicin E9

ABSTRACT Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9s-s) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9s-s, we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9AF) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.

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Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity

Virology ◽

10.1016/j.virol.2009.07.018 ◽

2009 ◽

Vol 393 (1) ◽

pp. 144-150 ◽

Cited By ~ 55

Author(s):

Lanying Du ◽

Guangyu Zhao ◽

Chris C.S. Chan ◽

Shihui Sun ◽

Min Chen ◽

...

Keyword(s):

Receptor Binding ◽

Protective Immunity ◽

Neutralizing Antibody ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

E Coli ◽

Recombinant Receptor

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Faculty Opinions recommendation of Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1728969.1246069 ◽

2010 ◽

Author(s):

Grant McFadden ◽

Kim Van Vliet

Keyword(s):

Receptor Binding ◽

Protective Immunity ◽

Neutralizing Antibody ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

E Coli ◽

Recombinant Receptor

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Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Aluminum Hydroxide Induces Protective Immunity and Reduces Immune Enhancement

10.1101/2020.05.15.098079 ◽

2020 ◽

Cited By ~ 15

Author(s):

Wen-Hsiang Chen ◽

Xinrong Tao ◽

Anurodh Agrawal ◽

Abdullah Algaissi ◽

Bi-Hung Peng ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Binding Domain ◽

Pseudotyped Virus ◽

Receptor Binding Domain ◽

Protein Vaccine ◽

S Protein ◽

Recombinant Receptor ◽

High Level

AbstractWe developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.

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A recombinant receptor-binding domain in trimeric form generates completely protective immunity against SARS-CoV-2 infection in nonhuman primates

10.1101/2021.03.30.437647 ◽

2021 ◽

Author(s):

Limin Yang ◽

Deyu Tian ◽

Jian-bao Han ◽

Wenhui Fan ◽

Yuan Zhang ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Nonhuman Primates ◽

Rhesus Macaques ◽

Vaccination Strategy ◽

Binding Domain ◽

Receptor Binding Domain ◽

Recombinant Receptor ◽

High Level ◽

Cellular Immune

Safe and effective vaccination is critical to combatting the COVID-19 pandemic. Here, we developed a trimeric SARS-CoV-2 receptor-binding domain (RBD) subunit vaccine candidate that simulates the natural structure of the spike (S) trimer glycoprotein. Immunization with RBD-trimer induced robust humoral and cellular immune responses and a high level of neutralizing antibodies that were maintained for at least 4 months. Moreover, the antibodies that were produced in response to the vaccine effectively neutralized the SARS-CoV-2 501Y.V2 variant. Of note, when the titers of the antibodies dropped to a sufficiently low level, only one boost quickly activated the anamnestic immune response, resulting in complete protection against the SARS-CoV-2 challenge in rhesus macaques without typical histopathological changes or viral replication in the lungs and other respiratory tissues. Our results indicated that immunization with SARS-CoV-2 RBD-trimer could raise long-term and broad immunity protection in nonhuman primates, thereby offering an optimal vaccination strategy against COVID-19.

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Expression, Purification, and in vivo Evaluation of GFP-Fused M Cell Targeting Receptor Binding Domain of Protein FimH

Protein and Peptide Letters ◽

10.2174/0929866526666190405122805 ◽

2019 ◽

Vol 26 (9) ◽

pp. 676-683 ◽

Cited By ~ 1

Author(s):

Thanh-Hoa Nguyen-Thi ◽

Kien-Quang Huynh ◽

Phuong-Linh Dinh-Thi ◽

Linh-Thuoc Tran ◽

Yong-Suk Jang ◽

...

Keyword(s):

Receptor Binding ◽

Fluorescent Protein ◽

Vaccine Development ◽

Oral Vaccine ◽

Binding Domain ◽

Soluble Form ◽

M Cells ◽

Receptor Binding Domain ◽

In Vivo Evaluation ◽

E Coli

Background: The number of oral vaccines is still limited due to many difficulties suffered in the intestinal environment, such as mucosal clearance, vast area, harsh conditions, deteriorative enzymes, impermeability, tolerance, etc. Numerous strategies have focused on directing antigen to the receptors of M cells, which is the main gateway to acquire and initiate specific responses to antigens in intestine. FimHrb is a receptor binding domain of type 1 of fimbriae from E. coli and Salmonella that can bind to GP2 receptor expressed exclusively on M cells. Objective: In this study, we evaluated the potential of FimHrb for oral vaccine development via its ability to adhere M cells. Methods: The coding gene of FimHrb fused Green Fluorescent Protein (GFP) was cloned and expressed intracellularly in E. coli host strain. The recombinant protein FimHrb-GFP was then purified by IMAC method through 6x His tag designed downstream of GFP. Finally, the purified protein was monitored its binding on murine M cells in Payer Patch region. Results: Following the methods mentioned above, the coding gene FimHrb-GFP was successfully cloned into vector pET22b and intracellularly expressed in soluble form at low temperature induction. The purity and the recovered yield of this protein were 90% and 20%, respectively. After that, the adhesion of FimHrb-GFP was monitored in murine small intestine, which showed that the protein bound to Peyer Patch region and did not restrict on M cells. Conclusion: With the present data, we revealed a candidate protein FimHrb targeted receptor on M cells for oral vaccine development and other factors in E. coli would supplement FimH to provide the specific invasion of these bacteria via M cells.

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