Study on site-specific biotinylation of goat IgG mediated by photoactivatable protein G-Avitag fusion protein
10.3969/j.issn.1000-484X.2025.07.035
- VernacularTitle:光敏蛋白G-Avitag融合蛋白介导生物素对山羊IgG特异性位点偶联标记研究
- Author:
Yue DU
1
;
Yumin ZHANG
1
;
Lan MA
1
;
Mengran LI
1
;
Hongming YANG
1
;
Jinbao TANG
1
Author Information
1. 山东第二医科大学药学院,潍坊 261053
- Publication Type:Journal Article
- Keywords:
Protein G;
Goat IgG;
Avitag;
Photoaffinity labeling
- From:
Chinese Journal of Immunology
2025;41(7):1777-1781
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct three photosensitive fusion proteins between protein G C3 domain and Avitag for site-spe-cific biotinylation of goat IgG.Methods:DNA sequences of protein G C3 domain with amber codon mutation and C-terminal containing 1,2,3 Avitag were obtained by chemical synthesis,respectively,and sub-cloned into pTXB1 plasmid to construct three fusion pro-tein expression vectors.Three plasmids and pEVOL-pBpF plasmid were co-transformed into competent bacterium E.coli BL21,respec-tively.Target fusion proteins CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 were prepared using genetic codon extension technology,and were purified by His Trap Chelating HP column,then biotinylated by Avitag/BirA system.Photo-conjugation efficiency,crosslink site and antigen-binding activity of photo-conjugates were investigated by SDS-PAGE,control experiment,and competitive ELISA.Results:Three expression vectors were successfully constructed,and three designed photoactivatable fusion proteins CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 were successfully expressed with molecular weights consistent with their theoretical values.Heavy-chain conjugation efficiencies of biotinylated fusion protein with goat IgG were 85%with Fc-specific conjugation,and conjugates maintained its antigen-binding activity after photo-conjugation.Conclusion:CBpa-Avitag,CBpa-(Avitag)2 and CBpa-(Avitag)3 fusion proteins are successfully prepared,and mediates by those photosensitive fusion proteins,biotins molecules are conjugated to Fc fragment of goat IgG,results site-specific biotinylation of goat IgG.
