title: Improving N-glycosylation efficiency in Escherichia coli using shotgun proteomics, metabolic network analysis, and selective reaction monitoring.
abstract: Recently, the prospect of using Escherichia coli as a host for human glycoprotein production has increased due to detailed characterization of the prokaryotic N-glycosylation process and the ability to transfer the system into this bacterium. Although functionality of the native Campylobacter jejuni N-glycosylation system in E. coli has been demonstrated, the efficiency of the process using the well-characterized C. jejuni glycoprotein AcrA, was found to be low at 13.4±0.9% of total extracted protein. A combined approach using isobaric labeling of peptides and probability-based network analysis of metabolic changes was applied to forward engineer E. coli to improve glycosylation efficiency of AcrA. Enhancing flux through the glyoxylate cycle was identified as a potential metabolic manipulation to improve modification efficiency and was achieved by increasing the expression of isocitrate lyase. While the overall recombinant protein titre did not change significantly, the amount of glycosylated protein increased by approximately 300%.
authors.affiliation: Department of Chemical and Biological Engineering, ChELSI Institute, Biological and Environmental Systems Group, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK.
authors.forename: Jagroop
authors.initials: J
authors.lastname: Pandhal
authors.forename: Saw Yen
authors.initials: SY
authors.lastname: Ow
authors.forename: Josselin
authors.initials: J
authors.lastname: Noirel
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D053858:Metabolic Networks and Pathways; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins