Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli

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.

Campylobacter jejuni Outer Membrane Vesicles Play an Important Role in Bacterial Interactions with Human Intestinal Epithelial Cells

“…The fact that Peb3 appears to be localized in the periplasm coupled with the reduction in the amount of Peb3 in pglB mutant OMVs compared to wild-type OMVs suggests that N-linked glycosylation may also play a role in OMV cargo selection. This is supported by the previous observation that when the C. jejuni N-linked glycosylation pathway is functionally transferred into E. coli, N-linked glycoproteins are identified within E. coli OMVs (20).…”

Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design

“…It was also demonstrated that the transient presence of the protein in the periplasm was sufficient for glycosylation to occur and glycoproteins could subsequently be trafficked to the outer membrane or even to the extracellular environment. This novel finding will be crucial for the engineering of future acceptor proteins for PGCT, particularly in the biosynthesis of novel glycoconjugate vaccines (Fisher et al, 2011).…”

“…For example, exotoxin A from P. aeruginosa is not naturally recognized by CjPglB but following the addition of two sequons, the modified protein can accept glycans and is now being tested as a glycoconjugate vaccine (Ihssen et al, 2010). Initially, it was considered that care had to be taken when selecting the grafting site in order not to interfere with the structural stability of the protein; however, it has been recently demonstrated that tags containing the ideal consensus sequence (glycotags) can be added to the N-and C-termini of proteins (Fisher et al, 2011). This means that it is not …”

“…Further analysis by nuclear magnetic resonance spectroscopy demonstrated that the structure of a glycosylatable loop of the native C. jejuni glycoprotein AcrA is very flexible (Slynko et al, 2009). Recent work by Fisher et al (2011) showed that adding a repeating 'tag' of optimal acceptor sequons to the N-or C-terminus of a protein is sufficient to render the protein a substrate for C. jejuni PglB (Fisher et al, 2011). This method was tested for up to eight sequons and for several different proteins, including a recombinant murine IgG protein, and all potential glycosylation sites were found to be occupied.…”

Increasing Recombinant Protein Production in E. coli by an Alternative Method to Reduce Acetate

“…In order to make E. coli produce N-linked glycoproteins the gene cluster pgl, responsible for glycosylation in Campylobacter jejuni (Szymanski et al, 1999;Abu-Qarn et al, 2008) was successfully transferred (Wacker et al, 2002). Moreover, combination of the pgl system with a simple, genetically encoded glycosylation tag, expands the glycosylation possibilities of E. coli (Fisher et al, 2011).…”