利用伴刀豆球蛋白检测O-甘露糖基化

目的:基于伴刀豆球蛋白A(ConA)特异性识别并结合甘露糖的特性,建立一种检测O-甘露糖基化的方法,为酵母等宿主表达蛋白的O-糖基化提供一种高效筛选和分析的方法。方法:利用糖苷酶F(PNGF)切除检测蛋白的N-糖链,排除N-糖基化的干扰;通过Q阴离子交换柱和ConA Sepharose 4B柱纯化Western印迹膜封闭蛋白牛血清白蛋白(BSA),除去BSA中甘露糖修饰的蛋白的干扰,优化膜封闭条件;利用辣根过氧化物酶标记的ConA检测具有低甘露糖型N-糖基化修饰能力的毕赤酵母GJK01-HL(Δoch1)表达的抗Her-2抗体是否存在O-甘露糖基化现象。结果:通过PNGF酶切处理,可以完全去除糖蛋白的N-糖链的干扰;BSA经过Q阴离子交换柱和ConASepharose 4B柱纯化后,除去了大部分甘露糖蛋白,可作为封闭蛋白;用建立的方法检测,发现毕赤酵母工程菌GJK01-HL(Δoch1)表达的抗Her-2抗体存在O-甘露糖基化现象。结论:本方法是研究糖蛋白是否发生O-甘露糖基化的有效检测手段,可用于酵母等表达蛋白的O-糖基化的高效筛选和分析。

Detection of the O-Mannosylation of Glycoproteins Using Lectin Concanavalin A

Objective： To develop a method for detecting the O-mannosylation of glycoproteins using lectin concanavalin A（ConA）,and for screening O-mannosylation modification of glycoproteins expressed in many organisms including yeast.Methods： PNGase F was used to remove N-glycans of the glycoprotein.Q colomn and ConA Sepharose 4B colomn were used to purify bovine serum albumin（BSA）.The O-mannosylation of anti-Her-2 monoclonal antibody expressed in Pichia pastoris mutant GJK01-HL（Δoch1） with high-mannosylation was detected by HRP-ConA.Results： The anti-Her-2 monoclonal antibody was modified with O-mannosylation while expressed in P.pastoris.Conclusion： The Western blotting combined with lectin ConA is a better method to detect the O-mannosylation modification of glycoproteins.It can be used in screening O-mannosylation of glycoproreins which they are expressed in many organisms including yeast.