Publication protocol
Selenomethionine(SeMet)-labeled EsxEFma, EsxGHms, and EsxOPmt complexes were expressed as previously described [26] in E. coli BL21 (DE3) with the following modifications: the EsxEFma and EsxGHms cultures were grown for 18 hours at 18°C after induction of protein expression with 0.5 mM IPTG while the EsxOPmt culture was grown under the same conditions but protein expression was induced with 1.0 mM IPTG. An unlabeled preparation of the EsxEFma complex was prepared using the same expression and purification conditions as for the SeMet-labeled EsxEFma complex with minor modifications as noted. The cells were harvested by centrifugation and the cell pellets were stored at −20°C pending lysis. Cell pellets were resuspended in lysis buffer (EsxEFma: 20 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol; EsxGHms and EsxOPmt: 50 mM HEPES, pH 7.8, 150 mM NaCl) containing protease inhibitor cocktail (Sigma), 2 mM β-mercaptoethanol, 1 mM PMSF, DNase I (0.5 µg/ml), and lysozyme. The cells were lysed by sonication, and the lysates clarified by centrifugation (30,000 x g for 30 minutes at 4°C). The supernatants were incubated with Ni-NTA agarose beads (Qiagen, Valencia, CA) for one to two hours at 4°C and the suspension was then poured into a gravity column. The beads were washed twice with wash buffer (lysis buffer with 10 mM imidazole and for EsxGHms 10% glycerol was included), once with high salt buffer (same composition as wash buffer but with 1 M NaCl), again with wash buffer containing 50 mM imidazole, and the complexes then eluted with elution buffer (same composition as wash buffer but with 0.3 M imidazole). The complexes were further purified by size exclusion chromatography using a HiPrep 16/60 Sephacryl S-100 column (GE Healthcare, Piscataway, NJ) equilibrated in: 20 mM Tris, pH 8.0, 0.3 M NaCl, 10% glycerol (SeMet-labeled EsxEFma), 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol (native EsxEFma), 20 mM HEPES, pH 7.8, 150 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol (EsxGHms), or 50 mM HEPES pH 7.8, 150 mM NaCl (EsxOPmt). The N-terminal His6 tag was cleaved from the native and SeMet-labeled EsxEFma complexes by TEV protease and the cleaved affinity tag and His6-tagged TEV protease separated from the cleaved target protein by passing the sample over Ni-NTA agarose beads prior to the size exclusion chromatography step. The SeMet-labeled EsxEFma complex was dialyzed into buffer IEX-A (20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol) and further purified by ion exchange chromatography using a HiTrap Q HP column (GE healthcare) using a linear gradient from 0–100% buffer IEX-B (20 mM Tris, pH 8.0, 500 mM NaCl, 10% glycerol). The pure SeMet-labeled EsxEFma complex was subsequently dialyzed against storage buffer (20 mM Tris, pH 8.0, 0.3 M NaCl, 10% glycerol). The purified complexes were then concentrated for crystallization screening [EsxEFma (native): 11 mg/ml; EsxEFma (SeMet-labeled): 4.5 mg/ml; EsxGHms: 27 mg/ml; EsxOPmt: 16 mg/ml].
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