title: Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis.
abstract: The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspI ATP binding domain, MtEspI415-493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. (31)P NMR titration assay indicated that MtEspI415-493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins.
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
authors.forename: Hanyu
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
authors.forename: Huilin
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
authors.forename: Shuangliang
authors.initials: S
authors.lastname: Tian
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
authors.forename: Donghai
authors.initials: D
authors.lastname: Lin
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China. Electronic address: guochy78@xmu.edu.cn.
authors.forename: Chenyun
authors.initials: C
authors.lastname: Guo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000942:Antigens, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; C000617171:EspI protein, Mycobacterium tuberculosis; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate
keywords: (31)P NMR titration; Bioinformatics analysis; MtEspI(415–493); Recombinant preparation