Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.
Appl Environ Microbiol
; 77(3): 871-81, 2011 Feb.
Article
em En
| MEDLINE
| ID: mdl-21131519
ABSTRACT
The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
/
Glicoproteínas
/
Engenharia de Proteínas
/
Escherichia coli
Idioma:
En
Revista:
Appl Environ Microbiol
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos