Transglutaminase-based antibody-drug conjugation: antibody site-specific mutation and identification / 药学学报
Acta Pharmaceutica Sinica
; (12): 403-408, 2017.
Article
em Zh
| WPRIM
| ID: wpr-779606
Biblioteca responsável:
WPRO
ABSTRACT
Transglutaminase (TG) posttranslational modification of antibody permits more precisely conjugating. Based on the amino acid sequence of an anti-CD24 antibody (cG7), this article is aimed to generate a deglycosylated cG7 mutant (cG7Q). Firstly, we introduced additional glutamines at position 297 (N297Q) by site-directed mutagenesis, and then transfected the recombinant plasmids into CHO-s cells via electroporation method and screened by Dot blot assay. Subsequently, cG7Q was expressed and purified through Protein A affinity chromatography, further identified by SDS-PAGE electrophoresis and Western blot. Its affinity was detected with surface plasmon resonance and flow cytometry assay, and ADCC effect was determined by lactate dehydrogenase (LDH) release. Eventually, a cG7 mutant, cG7Q was successfully expressed with sequence-specific conjugation sites for further study.
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1
Índice:
WPRIM
Tipo de estudo:
Diagnostic_studies
Idioma:
Zh
Revista:
Acta Pharmaceutica Sinica
Ano de publicação:
2017
Tipo de documento:
Article