National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shandong 250100, China.
Biochem Biophys Res Commun. 2010 Apr 16;394(4):1069-74. doi: 10.1016/j.bbrc.2010.03.126. Epub 2010 Mar 21.
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.
空肠弯曲菌含有一个翻译后 N-糖基化系统，其中 PglB 同源物作为寡糖基转移酶发挥作用。在此，我们建立了一种获得大量同质 PglB 蛋白的方法。PglB 在大肠杆菌 C43(DE3)中的表达水平达到 1mg/L 细胞培养物。使用化学合成的糖供体：N-乙酰半乳糖胺二磷酸十一碳烯基（GalNAc-PP-Und）和合成肽受体验证了纯化的 PglB 的活性。结果证实 PglB 仅负责寡糖基转移酶活性，并补充了 PglB 表现出宽松糖底物特异性的发现。此外，我们使用 PhoA/LacZ 融合方法对 PglB 进行了拓扑作图。拓扑模型表明，PglB 具有 11 个跨膜片段和两个相对较大的周质区域，除了 C 末端结构域外，这与 STT3 家族蛋白的常见 N(cyt)-C(peri)拓扑结构的 11 个跨膜片段的提议一致。
