Mansell Thomas J, Guarino Cassandra, DeLisa Matthew P
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
Biotechnol J. 2013 Dec;8(12):1445-51. doi: 10.1002/biot.201300026. Epub 2013 Oct 4.
Predicting the structural consequences of site-specific glycosylation remains a major challenge due in part to the lack of convenient experimental tools for rapidly determining how glycosylation influences protein folding. To address this shortcoming, we developed a genetic selection that directly links the in vivo folding of asparagine-linked (N-linked) glycoproteins with antibiotic resistance. Using this assay, we identified three known or putative glycoproteins from Campylobacter jejuni (Peb3, CjaA, and Cj0610c) whose folding was significantly affected by N-glycosylation. We also used the genetic selection to isolate a glycoengineered variant of the Escherichia coli colicin E7 immunity protein (Im7) whose intracellular folding and stability were enhanced as a result of N-glycosylation. In addition to monitoring the effect of glycan attachment on protein folding in living cells, this strategy could easily be extended for optimizing protein folding in vivo and engineering glycosylation enzymes, pathways, and hosts for optimal performance.
预测位点特异性糖基化的结构后果仍然是一项重大挑战，部分原因是缺乏便捷的实验工具来快速确定糖基化如何影响蛋白质折叠。为了解决这一缺陷，我们开发了一种遗传筛选方法，该方法将天冬酰胺连接的（N-连接的）糖蛋白的体内折叠与抗生素抗性直接联系起来。利用该检测方法，我们从空肠弯曲杆菌中鉴定出三种已知或推测的糖蛋白（Peb3、CjaA和Cj0610c），其折叠受到N-糖基化的显著影响。我们还利用该遗传筛选方法分离出一种大肠杆菌大肠杆菌素E7免疫蛋白（Im7）的糖基工程变体，其细胞内折叠和稳定性因N-糖基化而增强。除了监测聚糖附着对活细胞中蛋白质折叠的影响外，该策略还可以很容易地扩展用于优化体内蛋白质折叠以及工程化糖基化酶、途径和宿主以实现最佳性能。
