短小芽孢杆菌(Bacillus pumilus)糖基转移酶基因的克隆、序列分析及表达

为获得具有催化活性的糖基转移酶纯酶,本研究以短小芽孢杆菌基因组DNA为模板,利用简并PCR技术扩增到基因(GT-A)全长序列.该序列全长1 287bp、编码423个氨基酸,分子量约为49.2KD.经序列分析,该基因属于糖基转移酶基因.根据GT-A基因开放阅读框序列设计引物,构建了原核表达重组质粒GTA-pet28a,并在大肠杆菌BL21(DE3)中成功诱导出了一个约50kD的融合蛋白.纯化后测定其糖基转移酶活性,与37℃相比,80℃的反应温度其活性能提高3.4倍左右.研究结果表明,该酶是一种具有应用潜力的嗜高温糖基转移酶.

Cloning,Sequence Analysis and Prokaryotic Expression of the Glycosyltransferase Gene in Bacillus pumilus

To get purified glycosyltransferase with catalytic ability,a full length sequence of GT-A gene was amplified by PCR reaction with genomic DNA in Bacillus pumilus as the template.The 1 287 bp sequence encoded a protein of 423 amino acid with molecular weight of about 49.2 kD.Sequence analysis implied that the gene was a glycosyltransferase gene.Prokaryotic recombinant expression vector GTA-pet28a was constructed based on the ORF(open reading frame) of the glycosyltransferase gene and was expressed in Escherichia coli BL21(DE3).A recombinant protein of about 50 kD was produced in the inducible expression system.The activity of the enzyme at 80 ℃ was 3.4 times higher than that at 37 ℃.The results show that the glycosyltransferase is a thermophilic enzyme with potential applications.