title: Expression, purification and characterization of Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis.
abstract: The Esx-1 cluster encodes a special secretion system that is important for granuloma formation and virulence when Mycobacterium tuberculosis infects the host. As one of the 'core' genes in the cluster, Rv3880c gene codes an Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis (MtEspL). It has been reported that EspL had a strong influence on the secretion of other two virulence factors, EsxA and EspE. However, so far little is known about the tertiary structure and specific function of MtEspL due to the difficulty in preparing the high-quality protein. In this study, we tried several fusion tags and various expression conditions to recombinantly express MtEspL. Through a four-step purification procedure, ultimately, we successfully prepared the full-length MtEspL in Escherichia coli BL21 (DE3) with a purity of 98%. The yields of the purified MtEspL protein were 14 mg/L in Luria Bertani medium and 5.6 mg/L in M9 minimal medium, respectively. Biophysical experiments showed that MtEspL existed in a dimeric form. Moreover, the (1)H-(15)N HSQC spectrum recorded on MtEspL illustrates a favorable dispersion of the resonance peaks, indicating that the symmetric dimeric MtEspL adopted a well-folded structure and might be feasible to determine its solution structure by NMR spectroscopy. Moreover, we identified a strong DNA-binding ability of MtEspL with fluorescence quenching experiments. Our work lays the basis for further structural determination and functional exploration of MtEspL.
authors: Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
authors: Shuangliang
authors: S
authors: Tian
authors: Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
authors: Hanyu
authors: H
authors: Chen
authors: Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
authors: Tao
authors: T
authors: Sun
authors: Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
authors: Huilin
authors: H
authors: Wang
authors: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200438, China.
authors: Xuelian
authors: X
authors: Zhang
authors: Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
authors: Yan
authors: Y
authors: Liu
authors: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, 361005, China.
authors: Jinmei
authors: J
authors: Xia
authors: Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China. Electronic address: guochy78@xmu.edu.cn.
authors: Chenyun
authors: C
authors: Guo
authors: Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China. Electronic address: dhlin@xmu.edu.cn.
authors: Donghai
authors: D
authors: Lin
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins
keywords: Biophysical techniques; MtEspL; NMR; Protein-DNA interaction; Recombinant preparation