Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.
STAR Protoc
; 4(1): 101974, 2023 03 17.
Article
em En
| MEDLINE
| ID: mdl-36633947
ABSTRACT
Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020)1, Cioce et al. (2021)2, and Cioce et al. (2022)3.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
N-Acetilgalactosaminiltransferases
Limite:
Humans
Idioma:
En
Revista:
STAR Protoc
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Reino Unido