A filamentous phage display system for N-linked glycoproteins


Celik E. , Fisher A. C. , Guarino C., Mansell T. J. , DeLisa M. P.

PROTEIN SCIENCE, cilt.19, ss.2006-2013, 2010 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 19 Konu: 10
  • Basım Tarihi: 2010
  • Doi Numarası: 10.1002/pro.472
  • Dergi Adı: PROTEIN SCIENCE
  • Sayfa Sayıları: ss.2006-2013

Özet

We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X-1-N-X-2-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 x 105 per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the -2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype-phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.