Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection

Du, Jinyan; Wu, Gang; Chen, Quanyao; Yu, Chuanfei; Xu, Gang; Liu, Anhui; Wang, Lan

doi:10.1016/j.ab.2022.115034

Cited by 3 publications

(6 citation statements)

References 40 publications

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“…The results (Figure 5) show that the experimental pI values for ACE2 (pI 3.9, and pI 4.1) and RBD (pI 8.8 and pI 9.4 for wild type and omicron, respectively) are lower than the calculated pI 5.4 for ACE2 (Figure 3) and pI 9.2 and pI 9.7 for RBD (Table 1). The obtained pI 9.2 is also somewhat different from the pI 9.02 for RBD measured by capillary electrophoresis [57]. This discrepancy can be caused by: (a) the difference between electrostatic interactions in 3D-folded and unfolded polypeptide chains; (b) the additional electric charges (ionized amino acid residues) of the polypeptide fragments attached to the C end of the used recombinant polypeptide chains (Fc tag for ACE2 and His tag for RBD); and (c) posttranslational modifications which can occur during the syntheses of recombinant proteins in cancer cell culture.…”

Section: Isoelectric Points Of Unfolded Polypeptide Chainscontrasting

confidence: 82%

Omicron Coronavirus: pH-Dependent Electrostatic Potential and Energy of Association of Spike Protein to ACE2 Receptor

Hristova,

Zhivkov

2023

Viruses

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The association of the S-protein of the SARS-CoV-2 beta coronavirus to ACE2 receptors of the human epithelial cells determines its contagiousness and pathogenicity. We computed the pH-dependent electric potential on the surface of the interacting globular proteins and pH-dependent Gibbs free energy at the association of the wild-type strain and the omicron variant. The calculated isoelectric points of the ACE2 receptor (pI 5.4) and the S-protein in trimeric form (pI 7.3, wild type), (pI 7.8, omicron variant), experimentally verified by isoelectric focusing, show that at pH 6–7, the S1–ACE2 association is conditioned by electrostatic attraction of the oppositely charged receptor and viral protein. The comparison of the local electrostatic potentials of the omicron variant and the wild-type strain shows that the point mutations alter the electrostatic potential in a relatively small area on the surface of the receptor-binding domain (RBD) of the S1 subunit. The appearance of seven charge-changing point mutations in RBD (equivalent to three additional positive charges) leads to a stronger S1–ACE2 association at pH 5.5 (typical for the respiratory tract) and a weaker one at pH 7.4 (characteristic of the blood plasma); this reveals the reason for the higher contagiousness but lower pathogenicity of the omicron variant in comparison to the wild-type strain.

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Section: Isoelectric Points Of Unfolded Polypeptide Chainscontrasting

confidence: 82%

Omicron Coronavirus: pH-Dependent Electrostatic Potential and Energy of Association of Spike Protein to ACE2 Receptor

Hristova,

Zhivkov

2023

Viruses

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“…Monomeric RBD (319-541) from HEK-293 cells presents with a range of isoforms varying from 7.36 to 9.88 ( Krebs et al, 2021 ). Our RBD (319-541)-His6 dimer has a narrower range of isoforms compared to the trimeric RBD (319-537) expressed in CHO cells ( Du et al, 2023 ).…”

Section: Resultsmentioning

confidence: 89%

“…Although there is no reported isoelectric point for the RBD (319-541) dimer, studies have been conducted on the monomeric and trimeric charged variants using capillary isoelectric focusing ( Krebs et al, 2021 ; Du et al, 2023 ).…”

Section: Resultsmentioning

confidence: 99%

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens

Boggiano-Ayo,

Palacios-Oliva,

Lozada-Chang

et al. 2023

Front. Bioeng. Biotechnol.

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We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2 L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500 L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms.

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“…iCIEF was applied for the fingerprinting of the trimeric receptor binding domain (RBD) of the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) that is together with the whole S‐protein the target to be genetically engineered for designing the wide‐spectrum vaccine against SARS‐CoV‐2 [108]. The separation was performed in the fluorocarbon‐coated FS capillary column (50 mm long × 100/200 µm id/od) using 200 µL of the aqueous amphoteric electrolyte system composed of mixed ampholytes of 3 µL of Pharmalytes pH 3–10 and 1 µL of Pharmalytes pH 8–10.5, 10 µL of 200 mM iminodiacetic acid, 5 µL of arginine and 35 µL of 1% methylcellulose, and 1 µL of two p I markers (4.05 and 10.45).…”

Section: Separations By the Particular Ce Methodsmentioning

confidence: 99%

“…In addition to the separations in strongly acidic or alkaline BGEs or in BGEs with high ionic strength, various types of capillary and microchip coatings are used. Dynamic coatings are based on reversible (dynamic) adsorption of small ions, for example, oligoamines and detergents (SDS, CTAB), or hydrophilic polymers and cellulose derivatives, for example, methylcellulose [108] or hydroxypropylcellulose (HPC) [109] added to the BGE to the capillary or microchannel wall. Static (permanent) modifications of the inner capillary or microchannel wall include covalent bonding or strong physical adsorption of the synthetic and natural polymers, for example, neutral linear polyacrylamide (LPA) [110], PVA [111], poly(vinyl pyrrolidone) [92], poly(ethylene oxide) (PEO) [112], PEG [113], fluorocarbon [114], and linear and grafted cellulose derivatives [108,114], or charged polymers, such as cationic polybrene (PB) (hexadimethrine bromide) [94], poly(diallyldimethylammonium chloride) (PDADMAC) [113], and PEI [115], and anionic poly(sodium styrene sulfonate) (PSS) [113], and dextran sulfate [110].…”

Section: Suppression Of Peptide Adsorption and Regulation Of Eofmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

Kašička

2023

Electrophoresis

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This review article brings a comprehensive survey of developments and applications of high‐performance capillary and microchip electromigration methods (zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical characterization of peptides in the period from 2021 up to ca. the middle of 2023. Progress in the study of electromigration properties of peptides and various aspects of their analysis, such as sample preparation, adsorption suppression, electroosmotic flow regulation, and detection, are presented. New developments in the particular capillary electromigration methods are demonstrated, and several types of their applications are reported. They cover qualitative and quantitative analysis of synthetic or isolated peptides and determination of peptides in complex biomatrices, peptide profiling of biofluids and tissues, and monitoring of chemical and enzymatic reactions and physicochemical changes of peptides. They include also amino acid and sequence analysis of peptides, peptide mapping of proteins, separation of stereoisomers of peptides, and their chiral analyses. In addition, micropreparative separations and physicochemical characterization of peptides and their interactions with other (bio)molecules by the above CE methods are described.

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Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection

Cited by 3 publications

References 40 publications

Omicron Coronavirus: pH-Dependent Electrostatic Potential and Energy of Association of Spike Protein to ACE2 Receptor

Omicron Coronavirus: pH-Dependent Electrostatic Potential and Energy of Association of Spike Protein to ACE2 Receptor

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

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