Expression and purification of a jasmonic acid carboxyl methyltransferase from Salvia miltiorrhiza in E.coli
10.16438/j.0513-4870.2016-0414
- VernacularTitle:丹参茉莉酸甲基转移酶蛋白表达和纯化的研究
- Author:
Na YAO
1
;
Han ZHENG
1
;
Li JING
1
;
Li-gang MA
2
;
Ye SHEN
1
;
Min CHEN
1
Author Information
1. State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
2. Henan University of Traditional Chinese Medicine, Zhengzhou 450016, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
Salvia miltiorrhiza;
jasmonic acid carboxyl methyltransferase;
protein expression and purification;
Western blotting;
Q-TOF
- From:
Acta Pharmaceutica Sinica
2016;51(10):1643-
- CountryChina
- Language:Chinese
-
Abstract:
Jasmonic acid carboxyl methyltransferase (JMT), a key enzyme for jasmonate (JA) biosynthesis, catalyzes the methylation of JA to form MeJA. To characterize the function of JMT, a plasmid pGEX-4T-SmJMT1 harboring JMT1 (SmJMT1) gene from Salvia miltiorrhiza was successfully transformed into E.coli BL21 (DE3) for protein expression. The recombination SmJMT1 was separated using SDS-PAGE and the size of expressed SmJMT1 protein was consistent with the prediction. The bacterial growth conditions were determined for optimal expression, which include growth temperature, incubation time, IPTG concentrations and culture density. The optimal growth conditions for SmJMT1 were that the bacterial cultures were grown to an A600 of 0.8, and induced with IPTG at a final concentration of 0.4 mmol·L-1, and then incubated for 8 h at 20℃. The expression of SmJMT1 in E.coli was confirmed by Western blotting, and mass spectrometry analysis of methyltransferase family. The successful expression and purification of JMT in this study provide the basis for more study of JA biosynthetic pathway and JA-regulated secondary metabolism of medicinal plants.
