Engineered genetic selection links in vivo protein folding and stability with asparagine-linked glycosylation

Biotechnol J. 2013 Dec;8(12):1445-51. doi: 10.1002/biot.201300026. Epub 2013 Oct 4.


Predicting the structural consequences of site-specific glycosylation remains a major challenge due in part to the lack of convenient experimental tools for rapidly determining how glycosylation influences protein folding. To address this shortcoming, we developed a genetic selection that directly links the in vivo folding of asparagine-linked (N-linked) glycoproteins with antibiotic resistance. Using this assay, we identified three known or putative glycoproteins from Campylobacter jejuni (Peb3, CjaA, and Cj0610c) whose folding was significantly affected by N-glycosylation. We also used the genetic selection to isolate a glycoengineered variant of the Escherichia coli colicin E7 immunity protein (Im7) whose intracellular folding and stability were enhanced as a result of N-glycosylation. In addition to monitoring the effect of glycan attachment on protein folding in living cells, this strategy could easily be extended for optimizing protein folding in vivo and engineering glycosylation enzymes, pathways, and hosts for optimal performance.

Keywords: Bacterial glycosylation; Glycoengineering; Glycoprotein-folding reporter; Post-translational modification; Protein engineering.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Asparagine / chemistry
  • Asparagine / genetics
  • Asparagine / metabolism*
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Genetic Engineering / methods*
  • Glycoproteins* / chemistry
  • Glycoproteins* / genetics
  • Glycoproteins* / metabolism
  • Glycosylation
  • Protein Folding*
  • Protein Stability*


  • Carrier Proteins
  • E colicin-binding immunity protein Im7, E coli
  • Escherichia coli Proteins
  • Glycoproteins
  • Asparagine