SBIR TOPIC 269: AN EXPRESSION SYSTEM FOR SYNTHESIS OF GLYCOPROTEIN WITH DEFINED

粘蛋白型糖基化是最普遍的蛋白质翻译后修饰之一， 在许多生物过程中起重要作用。粘蛋白和粘蛋白样糖蛋白是潜在的癌症 标记。糖蛋白通常作为异质糖型从天然来源分离，并且这 使其结构和功能的研究复杂化，并阻碍了其亲和捕获的发展 试剂目前用于合成糖蛋白的方法耗时且技术要求高。 本项目的目标是建立一个简单、经济的无细胞翻译系统 产生具有确定的O-聚糖结构的糖蛋白。O-聚糖将通过以下方式并入蛋白质中 在所需位点的翻译抑制。该项目的主要目标是开发氨酰-tRNA 合成酶（阿尔斯），其能够活化携带结构上确定的0-聚糖的糖氨基酸。相 我将致力于构建突变体阿尔斯文库，并建立选择二氨基葡萄糖合成酶的方法。 酸第二阶段将继续努力，完善糖基氨基酸的筛选过程， 各种聚糖结构。筛选还将扩展至II期的三-或四-糖基氨基酸。这些 选择的合成酶将用于高产率的无细胞翻译系统，以产生所需的0- 糖蛋白在第一阶段，目标是开发一个E。大肠杆菌无细胞表达系统，能够 产生含有T抗原的蛋白质，产量高达0.1 mg/1 ml翻译反应。

Mucin-type glycosylation is one of the most prevalent posttranslational modifications of proteins and plays an important role in many biological processes. Mucin and mucin-like glycoproteins are potential cancer markers. Glycoproteins are usually isolated as heterogeneous glycoforms from natural sources, and this complicates studies of their structure and function and hinders the development of their affinity capturing reagents. Current methods for synthesis of glycoproteins are time consuming and technically demanding. The goal of this project is to establish a cell-free translation system that can simply and economically produce glycoproteins with defined O-glycan structures. O-Glycans will be incorporated into proteins through translational suppression at desired site(s). The main objective of the project is to develop amino acyl-tRNA synthetases (aaRS) that are able to activate glycoamino acids carrying structurally defined O-glycans. Phase I will focus on construction mutant aaRS libraries and establish methods for selecting synthetases for a diglycoamino acid. Phase II will continue the effort and refine the screen process for glycoamino acids with various glycan structures. The screen will also extend to tri- or tetra-glycoamino acid in Phase II. These selected synthetases will be used in high yield cell-free trans lation system to produce desired 0- glycoprotein. In phase I, the objective is to develop an E. coli cell-free expression system that is able to produce protein containing T -antigens in a yield up to 0.1 mg per 1 ml of translation reaction.