茶树咖啡碱合酶cDNA在大肠杆菌中的表达

Expression of tea caffeine synthase cDNA in Escherichia coli

通过RT PCR法扩增出茶树咖啡碱合酶 (TCS)cDNA编码区全序列 ,并将其克隆到表达载体pET 32a (+)中 ,得到重组质粒pET TCS ,在表达宿主菌BL2 1trxB (DE3)中高效表达 ,产生相对分子质量约为 6 2 0 0 0的融合蛋白。该融合蛋白包括 112个氨基酸残基的硫氧还原蛋白及 370个氨基酸残基的茶树TCS蛋白。实验结果表明 ,随着诱导时间的延长 ,表达的融合蛋白产量逐渐增加 ,表达蛋白总量最高可达到菌体总蛋白的 39 14 % ;在 15、 2 5和 37℃ 3个不同的诱导温度下 ,均能诱导产生融合蛋白 ,而且产生的总量及其可溶性部分所占比例均随诱导温度升高而增加 ;在菌体的各个部位中 ,融合蛋白主要在细胞质中以可溶的形式进行表达 ,有少量在外周质中表达或以包涵体形式在细胞质中表达。另外 ,体外酶促反应表明 ,表达的融合蛋白能催化可可碱甲基化生成咖啡碱 ,具有正常的生物学活性。

Tea caffeine synthase(TCS)gene cDNA code region was amplified by RT-PCR.The DNA fragment was cloned into expression vector pET 32a(+),forming recombinant pET-TCS.Expression of the plasmid in Escherichia coli BL21trxB(DE3)resulted in the production of a fusion protein(62 000)consisting of 112 amino acids of thioredoxin and 370 amino acids of TCS.With the inducing time increasing,the amount of fusion protein also increased,the highest content amounted to 39.14% of gross protein.Different temperature(15,25 and 37 ℃)could induce expression of fusion protein,and the higher temperature was,the more gross fusion protein and soluble fusion protein was produced.Most fusion protein was expressed in the cytoplasm and was soluble,a little of fusion protein was expressed in periplasm or expressed as inclusion bodies,and no fusion protein was found in culture.Furthermore,the results of in vitro enzymatic reaction suggested that the fusion protein had biological-activity and could catalyze the methylation of theobromine to caffeine.