The Escherichia coli glycophage display system

Abstract

We describe a phage display technique that allows the production and selective enrichment of phages that display an N-glycoprotein (glycophages). We applied glycophage display to select functional glycosylation sequons from a pool of randomized acceptor sequences. Our system provides a genetic platform to study and engineer different steps in the pathway of bacterial N-linked protein glycosylation.

Bacterial Protein N-Glycosylation: New Perspectives and Applications

“…C. jejuni PglB activity was either low, medium, or completely abolished in point mutants with substitutions of amino acid residues that are involved in substrate binding and catalysis, confirming and supporting the observations made by Lizak et al (82) 5 The fact that D475V and K478V single and double point mutants displayed a significant reduction in PglB-mediated free oligosaccharide release and N-glycosylation levels when analyzed in the native C. jejuni host suggests that these residues are indeed important for PglB activity. 5 The PglB structure in combination with in vitro evolution systems such as cell-surface and phage display (86,87) will lead to the creation of OTases that efficiently glycosylate eukaryotic acceptor sites. The identification and characterization of novel OTases (88), especially those with more relaxed substrate specificities (28,81), will assist in engineering novel antigens.…”

Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

“…We hypothesized that such variants could be isolated by laboratory evolution using a reporter assay that generates a genotype-glycophenotype linkage. A handful of genetic screens for N -linked glycosylation have been described for this purpose including ELISA-based detection of periplasmic glycoproteins 16, 17 , glycophage display 18, 19 , and cell surface display of glycoconjugates 20–22 . All of these involve the use of glycoengineered Escherichia coli carrying the complete protein glycosylation ( pgl ) locus of C. jejuni 23 ; however, none have been used to engineer OST variants with improved or novel activities.…”

Strain engineering for improved expression of recombinant proteins in bacteria

“…Several groups have started to utilize the C. jejuni pgl N -linked glycosylation platform for biotechnological applications, including the generation of glyco-conjugated vaccines in bacteria [44,45]. Very recently, two groups have reported the display of glycoproteins onto filamentous phage, which in turn may enable the isolation of novel types of glycoproteins from combinatorial libraries [46,47]. …”