Site-specific glycosylation of a functional recombinant protein thioester is reported. The thioester functionalized protein sfGFP-Y151ThioD, prepared by genetic code expansion, underwent native chemical ligation with the cysteine-conjugated glycans H-Cys-NH-GlcNAc and H-Cys-NH-(GlcNAc)₂(Man)₃ to give the corresponding cysteine-bridged glycoproteins. The intact glycoproteins, which retained their fluorescence, were characterized by top-down mass spectrometry and gel electrophoresis. The bridging cysteine provided a convenient handle for affinity chromatography purification of the glycoproteins via a removable biotin tag. Given the influence that specific glycoforms can have on a protein’s function, the ability to attach a homogeneous glycan to an intact protein in a functional group controlled yet sequon-independent manner could find widespread application. These preliminary results set the stage for development of the expressed protein glycoligation (EPG) concept.

中文翻译：

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半胱氨酸桥联糖蛋白通过表达的蛋白质糖基结合的位点特异性合成。

据报道功能性重组蛋白硫酯的位点特异性糖基化。通过遗传密码扩展制备的硫酯官能化蛋白sfGFP-Y151ThioD，与半胱氨酸偶联的聚糖H-Cys-NH-GlcNAc和H-Cys-NH-（GlcNAc）₂（Man）₃进行天然化学连接得到相应的半胱氨酸桥糖蛋白。完整的糖蛋白保留其荧光，通过自上而下的质谱和凝胶电泳进行表征。桥接的半胱氨酸为通过可移动的生物素标签进行糖蛋白的亲和层析纯化提供了方便的方法。考虑到特定糖型可能对蛋白质功能产生的影响，以功能基团控制但不依赖序列的方式将均质聚糖连接至完整蛋白质的能力可能会得到广泛应用。这些初步结果为表达蛋白乙二醇化（EPG）概念的发展奠定了基础。