title: Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E. coli.
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 pGEX-4T-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 SDS-PAGE 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.
authors.affiliation: Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, 116023, China, hechongbo@hotmail.com.
authors.forename: Chongbo
authors.initials: C
authors.lastname: He
authors.forename: Panhai
authors.initials: P
authors.lastname: Chen
authors.forename: Xianggang
authors.initials: X
authors.lastname: Gao
authors.forename: Lei
authors.initials: L
authors.lastname: Gao
authors.forename: Le
authors.initials: L
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D003386:Brachyura / Q000378:metabolism*; D002681:China; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D026461:Ecdysteroids / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D060829:Arthropod Proteins; D018076:DNA, Complementary; D026461:Ecdysteroids; D011993:Recombinant Fusion Proteins; D004247:DNA