Site-Specific Synthesis of Cysteine-Bridged Glycoproteins via Expressed Protein Glycoligation
Site-Specific Synthesis of Cysteine-Bridged Glycoproteins via Expressed Protein Glycoligation
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通过表达蛋白糖基化位点特异性合成半胱氨酸桥糖蛋白
DOI：
10.1021/acs.bioconjchem.0c00437
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发表时间：
2020
影响因子：
4.7
通讯作者：
Garner, Philip
中科院分区：
文献类型：
--
作者：
Holloran, Nicholas;Collins, Daniel;Rathnayake, Upendra;Zhang, Bixia;Koh, Minseob;Kang, ChulHee;Garner, Philip
报道了一种功能性重组蛋白硫酯的位点特异性糖基化。通过基因密码子扩增制备的硫酯功能化蛋白sfGFP-Y151 ThioD与半胱氨酸结合的聚糖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，其特征在于由上而下的质谱和凝胶电泳。桥连半胱氨酸通过可去除的生物素标签为糖蛋白的亲和色谱纯化提供了方便的手柄。考虑到特定糖型对蛋白质功能的影响，以官能团受控但不依赖于序列的方式将均质聚糖连接到完整蛋白质的能力可以得到广泛的应用。这些初步结果为表达蛋白糖基化（EPG）概念的发展奠定了基础。
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)3to 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.