空肠弯曲菌β1,3-半乳糖基转移酶(CgtB)突变体的设计及活性鉴定

Design and Activity Assay ofβ1,3-Galactosyltransferase(CgtB)MutanTSfrom Bacterium Campylobacter jejuni

来自空肠弯曲菌的β1,3-半乳糖基转移酶(CgtB)主要负责蛋白质O-连接糖基化的第二步延伸,在O-连接N-乙酰半乳糖胺(O-GalNAc)修饰后面添加一个半乳糖,目前CgtB已经被广泛应用于体外O-连接糖基化的合成。为了开发O-GalNAc修饰的识别工具,CgtB蛋白质用于突变体设计。在基因水平删除N端30个氨基酸及C端的30个氨基酸得到Cgt1,将Cgt1构建在pMal-c5x表达质粒上并通过大肠杆菌进行原核表达。可溶性的Cgt1经过麦芽糖结合蛋白(MBP)亲和层析柱进行纯化并利用糖结合实验进行活性分析。结果显示,大肠杆菌体系可以成功表达可溶性重组Cgt1蛋白质,相对分子质量约为70000,重组的Cgt1具备结合O-GalNAc修饰蛋白质的活性;针对不同的标准糖蛋白,Cgt1只识别含有O-GalNAc修饰的黏蛋白(mucin);对于复杂的细胞样品,Cgt1可以特异性识别细胞内的O-GalNAc修饰。该研究开发的识别O-GalNAc修饰工具,为进一步研究O-GalNAc修饰的功能奠定了基础。

β1,3-galactosyltransferase(CgtB)from bacterium Campylobacter jejuni is mainly responsible for the second elongation of proteins O-glycosylation by transferring a galactose to O-linked N-acetylgalacosamine(O-GalNAc).Presently,CgtB has been widely applied in the synthesis of O-glycoproteins.To develop a recognition tool for O-GalNAc modification,the CgtB protein was used for mutant design.Cgt1 was generated by deleting 30 amino acids from both the N-and C-terminal regions at the gene level.Cgt1 was constructed in pMal-c5x expression plasmid and expressed in the Escherichia coli.The soluble Cgt1 was purified by affinity chromatography with maltose binding protein(MBP)column and the activity assay was carried out by glycan binding activity.The resulTSsuggested that the soluble recombinant Cgt1 with the molecular weight of approximately 70000 could be successfully expressed in the system of Escherichia coli.The recombinant Cgt1 possessed the activity of binding O-GalNAc modified proteins.For different standard glycoproteins,Cgt1 only recognized mucin containing O-GalNAc modification.For complex cellular samples,Cgt1 could specifically recognize intracellular O-GalNAc modification.The developed recognition tool for O-GalNAc modification in this research will provide a foundation for further research on the function of O-GalNAc modification.