北柴胡糖基转移酶基因BcUGT1的表达分析及其原核表达与蛋白纯化

Expression analysis of glycosyltransferase BcUGT1 from Bupleurum chinense DC. and its expression in E. coli and the target protein purification

本研究分析了北柴胡糖基转移酶基因BcUGT1 ORF序列,预测了其蛋白三维结构,利用qRT-PCR分析了BcUGT1的茉莉酸甲酯诱导表达和组织表达特性。结果表明,BcUGT1可能参与柴胡皂苷生物合成。随后,构建了BcUGT1原核表达载体,成功诱导出目标蛋白,并进行了纯化。原核表达实验中共采用了3种载体:pRSET-A、pET-28a(+)和pET-30a(+),3种宿主菌:BL21(DE3)plysS、BL21A1和BL21-CodonPlus(DE3)-RIPL,进行了不同诱导条件的探索,包括不同诱导物(L-阿拉伯糖和IPTG)的不同浓度、不同诱导时间和温度,结果以pET-28a(+)和pET-30a(+)为载体,BL21-CodonPlus(DE3)-RIPL为宿主菌,0.5或1 mmol.L 1IPTG,16℃,20 h为条件,诱导出目标蛋白。利用PrepEase His标签蛋白纯化试剂盒,纯化获得了目标蛋白,为后续开展BcUGT1基因的功能研究奠定了基础。

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate （MeJA） induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a （＋） and pET-30a （＋）, and three isolates of E. coli, BL21 （DE3） plysS, BL21A1 and BL21-CodonPlus （DE3）-RIPL were used under different induction conditions, such as different concentrations and during times of inducers （L-arabinose and IPTG） and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol＇L-1 IPTG, 16 ℃, 20 h, target protein expressed in BL21-CodonPlus （DE3）-RIPL with pET-28a （＋） or pET-30a （＋） as vector. Using PrepEase R His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.