杜仲EuGT4基因原核表达及其编码蛋白纯化

Prokaryotic Expression and Encoded Protein Purification of EuGT4 Gene in Eucommia ulmoides

为进一步研究杜仲糖基转移酶结构和功能提供科学依据,利用贵州省农业生物工程重点实验室构建的杜仲(Eucommia ulmoides Oliver)基因组数据库注释信息,从杜仲克隆得到1个全长为1 461bp的糖基转移酶(glycosyltransferase,GT)cDNA序列,将该基因命名为EuGT4;利用基因重组技术构建原核表达载体pET30a-EuGT4,遗传转化大肠杆菌BL21(DE3);在37℃条件下,以终浓度为0.2mM的异丙基β-D-硫代半乳糖苷(IPTG)诱导16h,并采用镍-亚氨基二乙酸(Ni-IDA)亲和层析柱纯化蛋白,以15%SDSPAGE凝胶电泳定性分析。结果表明:EuGT4重组蛋白以包涵体形式在BL21(DE3)中表达,经SDS-PAGE凝胶电泳检测和Western Blot鉴定,在相对分子质量约50kD处有1条特异性蛋白条带,确定为杜仲EuGT4蛋白。

To provide scientific basis for further research on the function and structure of glucosyltransferase in Eucommia ulmoides,aglycosyltransferase(GT)cDNA sequence with a total length of 1461 bp cloned fromE.ulmoides based on the annotation information of E.ulmoides genome database established by Guizhou Key Laboratory of Agricultural Bioengineeringis named as EuGT4.Prokaryotic expression vector pET30 a-EuGT4 is constructed by using gene recombination technology and genetically transforms into E.coli BL21(DE3).The EuGT4 recombinant protein is induced with 0.2 mM final concentration of isopropylβ-D-thiogalactoside(IPTG)at 37 ℃ for 16 h and purified with nickeliminodiacetic acid(Ni-IDA)affinity chromatography column.The purified protein is qualitatively analyzed by gel electrophoresis with 15% SDS-PAGE.Result:The EuGT4 recombinant protein expresses in BL21(DE3)in form of inclusion body.One specific protein band around 50 kD relative molecular mass is determined through SDS-PAGE gel electrophoresis detection and Western Blot identification,which indicates that EuGT4 recombinant protein is identified as EuGT4 protein in E.ulmoides.