Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E . coli
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Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E. coli Chongbo He • Panhai Chen • Xianggang Gao Lei Gao • Le Li
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Received: 8 October 2012 / Accepted: 17 October 2013 / Published online: 1 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. The goal of this study was to clone, efficiently express and purify the ecdysteroid-regulated protein (ERP) in the form of a GST fusion protein. The mature peptide-coding cDNA fragment was extracted from Chinese mitten crap (Eriocheir sinensis), and then after using PCR to obtain the open reading frame, a recombinant plasmid designated pGEX4T-1_ERP was successfully generated and showed to efficiently express the ERP fusion protein as determined by SDS-PAGE. The resulting expressed protein was successfully purified by a combination of affinity and conventional chromatographic methods. After purification, the recombinant protein showed the expected size of 41 kDa on SDSPAGE gels which was further confirmed by mass spectrometry and western blotting. Purification of recombinant protein was achieved by fast protein liquid chromatography. About 2.4 mg/l recombinant protein with purity more than 80 % was obtained. Keywords Chinese mitten crap (Eriocheir sinensis) Ecdysteroid-regulated protein Glutathione S-transferase Fusion expression E. coli
C. He (&) X. Gao L. Gao Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China e-mail: [email protected] C. He P. Chen L. Li College of Life Science, Liaoning Normal University, Dalian 116081, China P. Chen Wuxi Biortus Biosciences, Jiangyin 214437, China
Introduction Chinese mitten crab (Eriocheir sinensis) (Henri Milne Edwards 1854) is a unique species of crab that is native to China. It is a catadromous crustacean with a life span of *2 years. The crab enters the reproductive season in its second year and dies shortly after completing reproduction [1–5]. The crap grows by periodic molting, which is controlled by the molt-inhibiting hormone and ecdysteroid [6]. Ecdysteroid plays an important role in blood glucose regulation and regulates several important physiological processes including growth and reproduction [7]. So recently, the ecdysteroid-regulated gene which controlled the ecdysteroid has been widely noted, Cho found that the ecdysteroid-regulated gene cascades have important roles in oogenesis in insects [8–12]. And in 2001, Hepperle and Hartfelder [13] detected ecdysteroid-regulated gene expression during a critical phase of caste development in the honey bee. Then shock experiments revealed that in the honey bee ovary the 29 kDa/PI 4.6 ecdysteroid-regulated protein (ERP) belongs to the class of small heat shock proteins. And Lee [14] found that ERP was also an important element of crustacean metamorphos
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