N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Vaccine Development

Background:

• Site-specific protein modifications are crucial for biopharmaceutical development.
• Gluconoylation is a non-enzymatic modification of N-terminal HisTags.
• Current methods may lack efficiency or specificity.

Purpose of the Study:

• To report a high-yield, site-selective in vitro method for protein modification using azidogluconolactone.
• To demonstrate the application of this method in creating a multimeric antigen display for vaccine development.
• To evaluate the immunogenicity and efficacy of the developed vaccine candidate.

Main Methods:

• Site-selective α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5.
• Inhibition of conjugate hydrolysis using diol-masking with borate esters.
• Multimerization of azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry.

Main Results:

• High-yield and site-selective conjugation achieved within 1 hour.
• Demonstrated successful multimerization of RBD onto VLPs for a COVID-19 vaccine candidate.
• HEK-derived RBD showed superior immunogenicity compared to yeast antigen, attributed to glycosylation differences.
• Ordered multimeric antigen display resulted in single-shot seroconversion and potent virus-neutralizing antibodies.

Conclusions:

• Azidogluconoylation is a simple, fast, and robust chemistry for site-specific protein modification.
• This method accelerates research and development in biopharmaceuticals and vaccine design.
• Ordered antigen display on VLPs enhances immunogenicity and efficacy.