Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 419, Issue 3, Pages 472-476Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2012.02.020
Keywords
Glycosylation; Escherichia coli; Efficiency; Proteomics; Selective reaction monitoring
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Funding
- UK's Biotechnology and Biological Sciences Research Council (BBSRC) through the Bioprocess Research Industry Club (BRIC) [BBF0048421]
- Research Experience Placements (REP) scheme
- Biotechnology and Biological Sciences Research Council [BB/F004842/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/E036252/1] Funding Source: researchfish
- BBSRC [BB/F004842/1] Funding Source: UKRI
- EPSRC [EP/E036252/1] Funding Source: UKRI
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Recently, efforts to increase the toolkit which Escherichia coli cells possess for recombinant protein production in industrial applications, has led to steady progress towards making glycosylated therapeutic proteins. Although the desire to make therapeutically relevant complex proteins with elaborate human-type glycans is a major goal, the relatively poor efficiency of the N-glycosylation process of foreign proteins in E. coli remains a hindrance for industry take-up. In this study, a systematic approach was used to increase glycoprotein production titres of an exemplar protein, AcrA, and the resulting glycosylation efficiency was quantified using a combination of Western blots and pseudo Selective Reaction Monitoring (pSRM). Western blot and pSRM results demonstrate that codon optimising the oligosaccharyltransferase, PglB, for E. coli expression, increases efficiency by 77% and 101%, respectively. Furthermore, increasing expression of glycosyltransferase, WecA, in E. coli improves efficiency by 43% and 27%, respectively. However, increasing the amount of donor lipid used in the glycosylation process did not impact on the glycosylation efficiency in this system, with this specific protein. (C) 2012 Elsevier Inc. All rights reserved.
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