Overexpression and topology of bacterial oligosaccharyltransferase PglB

• Published in: Biochemical and biophysical research communications 2010-04, Vol.394 (4), p.1069-1074
• Main Authors: Li, Lei, Woodward, Robert, Ding, Yan, Liu, Xian-wei, Yi, Wen, Bhatt, Veer S., Chen, Min, Zhang, Lian-wen, Wang, Peng George
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; Amino Acid Sequence; Campylobacter jejuni; Campylobacter jejuni - enzymology; Campylobacter jejuni - genetics; COMPLEMENT; DICHROISM; ESCHERICHIA COLI; Escherichia coli - genetics; Galactosamine - analogs & derivatives; Galactosamine - chemistry; GLUCOSE; Glycosylation; Hexosyltransferases - chemistry; Hexosyltransferases - genetics; Hexosyltransferases - metabolism; L CELLS; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Sequence Data; N-glycosylation; Oligosaccharyltransferase; Overexpression; PEPTIDES; Polyisoprenyl Phosphates - chemistry; Protein Conformation; Protein Processing, Post-Translational; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; SACCHAROSE; SUBSTRATES; Topology
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2010.03.126

Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common N cyt–C peri topology with 11 transmembrane segments for the STT3 family proteins.