HBV相关糖基转移酶Glt25D2的核苷酸单糖结合活性分析

Assay of the nucleotide sugar binding activity of Glt25D2,a glycosyltransferase associated with HBV

目的体外克隆表达人类糖基转移酶Glt25D2,利用Biacore分析系统对其活化单糖及钙离子结合活性进行分析。方法构建Glt25D2原核表达载体pET32a-Glt25D2,转化大肠埃希菌BL21,克隆表达糖基转移酶Glt25D2。离心集菌,制备蛋白样品,进行SDS-PAGE电泳分析;融合蛋白线性梯度洗脱,Ni-NTA柱纯化,后做Western blot鉴定。用纯化后的Glt25D2融合蛋白包被CM5芯片,分别用不同浓度的活性单糖及钙离子灌注芯片,利用Biacore生物分子相互作用分析仪分析Glt25D2的活化单糖及钙离子结合活性。结果体外成功表达人类糖基转移酶Glt25D2融合蛋白。Biacore分析显示,该糖基转移酶与400、200、100、50μg/ml唾液酸的结合活性分别为108、71、50和20 RU,与200、100、50、0μg/ml钙离子的结合活性分别为37、20、10和0 RU。结论人类糖基转移酶Glt25D2重组蛋白具有较强的钙离子及唾液酸结合活性。

Objective To express the human glycosyltransferase Glt25D2 and analyze it using Biacore.Methods pET32a-Glt25D2,a prokaryotic expression vector of Glt25D2,was constructed and transformed into BL21 to express Glt25D2.SDS-PAGE and Western blotting were used to evaluate purified Glt25D2 fusion protein.Biacore was used to analyze the binding activity of Glt25D2 with nucleoside sugar and Ca2＋.Results Glt25D2 fusion protein was successfully expressed.Biacore results showed that Glt25D2 had strong binding activity with sialic acid and Ca2＋.Conclusion Prokaryotically expressed Glt25D2 had strong binding activity with sialic acid and Ca2＋.