OjCHS原核表达载体的构建及重组蛋白的纯化

查尔酮合酶（chalcone synthase，CHS）是类黄酮生物合成过程中的第一个关键酶，也是限速酶，它直接影响并限制类黄酮的合成与积累。在克隆得到日本蛇根草CHS基因（OjCHS）基础上，将该基因插入原核表达载体pET-28a，完成重组表达质粒pET28a-CHS的构建。随后，利用冻融法将上述质粒转入大肠杆菌BL21感受态细胞中用于重组蛋白的表达。选择不同IPTG 浓度、诱导温度及诱导时间对重组菌进行诱导，确定了可溶性重组蛋白的最佳诱导表达条件。最后，通过洗脱、纯化、透析与浓缩完成重组蛋白的纯化。结果显示，成功构建原核表达载体pET28a-CHS，可溶性重组蛋白最佳诱导条件为：IPTG 浓度0.45 mmol·L^-1，25 ℃下诱导12 h。最后大量制备并纯化获得符合预期大小的可溶性蛋白，为深入研究OjCHS的生物学功能奠定了基础。

Prokaryotic expression vector construction and recombinant proteins purification of OjCHS

Chalcone synthase (CHS) is the first and rate-limiting enzyme in the biosynthesis of flavonoids, and also plays important role during the biosynthesis and accumulation of flavonoids. In this study, we inserted OjCHS into pET-28a vector to obtain recombinant pET28-CHS on the basis of the sequence of OjCHS. Then the recombinant plasmid was transfected into E.coli competent cell BL21 using a freeze-thaw method for the expression of recombinant proteins. Subsequently, the optimally induced expression condition was confirmed including IPTG concentration, inducing temperature and inducing time. Then a large number of soluble recombinant proteins were obtained through eluting with Ni column purification, further dialysis and concentration. The results showed that recombinant pET28-CHS vector was constructed and the optimally induced expression condition was 25 ^oC for 12 h by 0.45 mmol·L^-1 IPTG induction. Finally, we obtained abundant soluble recombinant protein which was consistent with the expected size, which would lay a foundation for the further study of the biological functions of OjCHS.