Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.
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.
Glycobia, Inc., 33 Thornwood Drive, Ithaca, New York 14850, USA.
Adam C
AC
Fisher
Charles H
CH
Haitjema
Cassandra
C
Guarino
Eda
E
Çelik
Christine E
CE
Endicott
Craig A
CA
Reading
Judith H
JH
Merritt
A Celeste
AC
Ptak
Sheng
S
Zhang
Matthew P
MP
DeLisa
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D019897:Periplasm / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D018271:Signal Recognition Particle