Production of initial-stage eukaryotic N-glycan and its protein glycosylation in Escherichia coli

Access this Article

Search this Article

Author(s)

Abstract

N-Glycosylation is a ubiquitous protein post-translational modification mechanism in eukaryotes. In this work, a synthetic pathway containing glycosyltransferases from Saccharomyces cerevisiae was introduced to Escherichia coli to synthesize lipid-linked mannosyl-chitobiose (Man-GlcNAc_2) and trimannosyl-chitobiose (Man_3-GlcNAc_2). Transfer of Man_3-GlcNAc_2 onto a model periplasmic protein occurred in the engineered E. coli cell using oligosaccharyltransferase PglB from Campylobacter jejuni. Mass spectrometric analysis of the fluorescently labeled N-glycan indicated a glycan signal composed of 2 HexNAc and 3 Hex residues. The reversed-phase HPLC analysis suggested that the Hex residues were α1,3-, α1,6- and β1,4-linked mannoses. These results indicated that the constructed system synthesizes a Man_3-GlcNAc_2, identical to that observed in an early eukaryotic dolichol pathway. Finally, glycopeptide mass spectrometry confirmed the transfer of the assembled glycan moiety onto an engineered glycosylation motif of recombinant maltose binding protein. Surprisingly, the Man_3-GlcNAc_2 structure but not Man-GlcNAc_2 was transferred onto maltose binding protein. This work showed that PglB protein might be able to accommodate the transfer of the further engineered glycan with greater complexity.

Journal

  • Journal of bioscience and bioengineering

    Journal of bioscience and bioengineering 119(4), 399-405, 2015-04

    The Society for Biotechnology, Japan

Codes

  • NII Article ID (NAID)
    110009942818
  • NII NACSIS-CAT ID (NCID)
    AA11307678
  • Text Lang
    ENG
  • ISSN
    1389-1723
  • NDL Article ID
    026336261
  • NDL Call No.
    Z53-S65
  • Data Source
    NDL  NII-ELS  Crossref 
Page Top