万古霉素糖基转移酶GtfE的异源表达与体外活性检测

基金项目: 国家自然科学基金资助项目(No.81072557,No.81172962)；国家“重大新药创制”科技重大专项资助项目(No.2012ZX09201101-008)

Heterologous expression and in vitro activity of vancomycin glycosyltransferase GtfE

摘要: 为了得到具有糖基转移酶活性的重组蛋白，从万古霉素产生菌总DNA中克隆万古霉素糖基转移酶基因gtfE，连入pET-37b构建表达质粒并转入大肠杆菌BL21(DE3)表达，体外测定纯化后的重组蛋白糖基转移酶活性，用HPLC和ESI-MS检测反应产物。结果表明，重组蛋白具有预期活性，可以催化尿苷二磷酸葡萄糖与万古霉素苷元的糖基化反应。本研究为获得糖基多样性的万古霉素奠定理论基础。
- 万古霉素 /
- 生物合成 /
- 糖基转移酶 /
- 异源表达 /
- 体外转糖反应
Abstract: Recombinant protein of vancomycin glycosyltransferase, GtfE, was obtained by gene cloning and hete-rologous expression. Gene gtfE was amplified from the genomic DNA of vancomycin producing strain and ligated into expression vector pET-37b, the recombinant plasmid GtfE/pET-37b was transformed into E. coli BL21(DE3). The product of in vitro glucosylation reaction catalyzed by purified GtfE was isolated and identified by HPLC and ESI-MS, respectively. The assay indicated that the recombinant protein GtfE had expected glucosylation activity to transfer uridine 5′-diphosphoglucose to the vancomycin aglycone. This study laid the foundation for producing glycosyl diversity of vancomycin.
- vancomycin /
- biosynthesis /
- glycosyltransferase /
- heterologous expression /
- in vitro glycosylation reaction