摘要
采用RT-PCR方法克隆拟南芥硫代葡糖苷酶基因TGG1,利用毕氏酵母表达。重组蛋白用镍亲和柱纯化,获得高纯度芥子酶。重组蛋白分子量78kD,与天然TGG1分子量接近,为糖基化蛋白。TGG1信号肽在酵母中具有分泌功能,约25%芥子酶分泌到培养基中。TGG1重组蛋白具有广泛的pH适应性,最适反应温度40℃左右。其活性被低浓度Vc激活,被高浓度Vc抑制。用Hanes plot方法计算TGG1重组蛋白,以Sinigrin为底物时,Km为65μmol/L,最大反应速率Vmax为3.28μmol/(min·mg)。
Myrosinase gene TGG 1 of Arabidopsis thaliana was cloned by RT-PCR and over expressed in Pichia pastoris.The recombinant protein of TGG 1 was purified with Ni-NTA.The recombinant protein had a molecular weight of 78 kD,larger than the deduced naked protein （58 kD）,but similar to the molecular weight of the natural TGG 1 protein.The TGG 1 signal peptide has some secretion function in yeast,resulted in approximately 25% myrosinases in the culture medium.The TGG 1 recombinant protein was active in a wide pH and temperature range.The optimal reaction temperature was around 40℃,and the pH between 6 and 9.Myrosinase activity was activated by low concentrations of ascorbic acid,and suppressed by high concentrations.The apparent K m and V max were 65 μmol/L and 3.28 μmol/（min·mg）,respectively when sinigrin was the substrate.
出处
《中国农学通报》
CSCD
北大核心
2010年第21期54-58,共5页
Chinese Agricultural Science Bulletin
基金
国家自然科学基金项目"新型芥子酶基因TGG4和TGG5的表达调控及生物学功能研究"(30571064)
关键词
拟南芥
芥子酶
硫代葡糖苷
重组蛋白
Arabidopsis thaliana
myrosinase
glucosinolate
recombinant protein
