茶树花青素还原酶基因在大肠杆菌中的表达及优化

茶树花青素还原酶是催化非酯型儿茶素EC和EGC合成的关键酶。采用RT-PCR技术,获得了茶树花青素还原酶基因的开放阅读框,它编码含337个氨基酸的蛋白质,推测分子量为37kD,等电点为6.54;成功地将该基因重组到表达载体pET32a(+)上,并在大肠杆菌rosetta中进行原核表达;优化了原核表达中诱导时间、诱导温度、IPTG浓度、氨苄青霉素浓度,纯化出目的蛋白。HPLC检测表明,目的蛋白具有ANR酶活性。

Expression and Optimization of Anthocyanin Reductase Gene of Tea Plant [Camellia sinensis (L.) O.Kuntze] in Escherichia coli

Anthocyanin reductase is a key enzyme in the biosynthesis of EC and EGC in tea plant.The open reading frame of anthocyanin reductase gene(ANR),which encoding a 337 amino acids protein,was cloned from tea plant(Camellia sinensis(L.) O.Kuntze) by RT-PCR.The deduced protein molecular weight was 37 kD and its theoretical isoelectric point was 6.54.The gene was cloned into the expression vector pET32a(+) for expression in prokaryotic cells.The SDS-PAGE results showed that the anthocyanin reductase peoteins was expressed in Escherichia coli rosetta.The optimal inducing conditions including time,temperature,IPTG concentration,ampicillin concentration were studied.The deduced protein was purified and its activity was detected by HPLC.