A filamentous phage display system for N-linked glycoproteins
A filamentous phage display system for N-linked glycoproteins
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DOI：
10.1002/pro.472
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发表时间：
2010-10-01
期刊：
影响因子：
8
通讯作者：
DeLisa, Matthew P.
中科院分区：
文献类型：
--
作者：
Celik, Eda;Fisher, Adam C.;DeLisa, Matthew P.
我们开发了一种丝状噬菌体展示系统，用于检测大肠杆菌中天门酰胺连接的糖蛋白，该系统携带编码空肠弯曲杆菌蛋白糖基化位点（pgl）的质粒。在我们的实验中，靶糖蛋白与次要噬菌体外壳蛋白g3p的融合导致了糖蛋白在噬菌体上的展示。在噬菌体上显示的糖表位是由空肠C. pgl途径编码的生物合成酶的产物，最低需要三个基本因素：低聚糖生物合成途径、功能性低聚糖转移酶和具有D/E-X-1-N-X-2-S/T基序的受体蛋白。凝集素层析可回收糖基化噬菌体，富集因子高达2 × 105 /轮，富集后的噬菌体在筛选后仍保持其传染性。通过这种实验，我们发现，通过在凝集素上筛选噬菌体展示的糖蛋白文库，可以可靠地选择所需的糖表型。例如，我们使用噬菌体选择来确定细菌共识受体位点序列-2位置的允许残基。综上所述，我们的研究结果表明，噬菌体相关糖表位和噬菌体编码基因之间可以建立基因型-表型联系，用于糖基化过程的三个基本组成部分中的任何一个。因此，我们预计我们的噬菌体展示系统可用于在糖基化过程的任何步骤中分离感兴趣的变异，从而使其成为大肠杆菌细胞中蛋白质糖基化遗传分析和糖工程的宝贵工具。
We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X-1-N-X-2-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 x 105 per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the -2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype-phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.