Overexpression and topology of bacterial oligosaccharyltransferase PglB |
| |
Authors: | Lei Li Robert Woodward Xian-wei Liu Veer S Bhatt Lian-wen Zhang |
| |
Institution: | a National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shandong 250100, China b Departments of Biochemistry and Chemistry, The Ohio State University, Columbus, OH 43210, USA c College of Pharmacy, Nankai University, Tianjin 300071, China |
| |
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 Ncyt-Cperi topology with 11 transmembrane segments for the STT3 family proteins. |
| |
Keywords: | OST oligosaccharyltransferase GalNAc N-acetylgalactosamine GlcNAc N-acetylglucosamine Glc glucose GalNAc-PP-Und N-acetylgalactosamine-diphospho-undecaprenyl IPTG d-galactospyranoside" target="_blank">isopropyl 1-thio-β-d-galactospyranoside CD circular dichroism |
本文献已被 ScienceDirect 等数据库收录! |
|