酵母中表达的糖蛋白片段的位点特异性化学修饰。
Site-specific chemical modification of a glycoprotein fragment expressed in yeast.
作者信息
Xiao Junpeng, Tolbert Thomas J
出版信息
Methods Mol Biol. 2011;751:329-42. doi: 10.1007/978-1-61779-151-2_21.
Abstract
Site-specific modification of glycoproteins has wide application in both biochemical and biophysical studies. This method describes the conjugation of synthetic molecules to the N-terminus of a glycoprotein fragment, viz., human immunoglobulin G subclass 1 fragment crystallizable (IgG1 Fc), by native chemical ligation. The glycosylated IgG1 Fc is expressed in a glycosylation-deficient yeast strain. The N-terminal cysteine is generated by the endogenous yeast protease Kex2 in the yeast secretory pathway. The N-terminal cysteine is then conjugated with a biotin thioester to produce a biotinylated, glycosylated IgG1 Fc using native chemical ligation.
摘要
糖蛋白的位点特异性修饰在生化和生物物理研究中都有广泛应用。本方法描述了通过天然化学连接将合成分子与糖蛋白片段（即人免疫球蛋白G亚类1可结晶片段（IgG1 Fc））的N端进行偶联。糖基化的IgG1 Fc在糖基化缺陷型酵母菌株中表达。N端半胱氨酸由酵母分泌途径中的内源性酵母蛋白酶Kex2产生。然后，利用天然化学连接将N端半胱氨酸与生物素硫酯偶联，以产生生物素化的、糖基化的IgG1 Fc。
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