Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells.
Protein Expr Purif
; 108: 23-29, 2015 Apr.
Article
em En
| MEDLINE
| ID: mdl-25527037
ABSTRACT
We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Glicoproteínas
/
Expressão Gênica
/
Glicogênio Sintase
/
Glucosiltransferases
/
Complexos Multienzimáticos
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Suíça