Glyco-Phage Display System for N-Linked Glycoproteins

Asparagine-linked (N-linked) protein glycosylation, an essential and conserved post-translational modification of diverse proteins in eukaryotic organisms, was found to occur also in archea and more recently in the bacterium Campylobacter jejuni. The glycosylation machinery that is encoded by a single locus (pgl) in C. jejuni has been functionally transferred into Escherichia coli, enabling the production of several N-glycosylated proteins of prokaryotic and eukaryotic origin in this well-known bacterial system. However, genetic analysis of the glycosylation pathway has been slowed by the lack of a reliable high-throughput screen or selection to rapidly interrogate N-linked glycosylation in these cells or to isolate mutants that confer desired N-glycosylation phenotypes.

Phage display is one of the most useful methods for isolating interesting mutants from large combinatorial libraries of protein sequences. We have extended the power of phage display for the production and selective enrichment of phages that display asparagine-linked (N-linked) glycoproteins. By displaying N-linked glycoproteins on M13 phage particles, a genotype-to-phenotype link is created between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Indeed, the desired glyco-phenotypes were reliably selected by panning phage-displayed glycoprotein libraries using lectin chromatography. The developed assay has significant potential for studying and engineering any of the steps along the bacterial N-linked protein glycosylation pathway.