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Overexpression and topology of bacterial oligosaccharyltransferase PglB. | LitMetric

Overexpression and topology of bacterial oligosaccharyltransferase PglB.

Biochem Biophys Res Commun

National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shandong 250100, China.

Published: April 2010

AI Article Synopsis

Article Abstract

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.

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Source
http://dx.doi.org/10.1016/j.bbrc.2010.03.126DOI Listing

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