Publication protocol
Expression and solubility of affinity tagged Esx complexes and their ability to bind Ni-NTA agarose was evaluated in 96 well format using a variation of the protocol of Klock et al. [25]. Briefly, E. coli BL21 (DE3) and/or E. coli Rosetta (DE3) harboring the expression plasmids were grown overnight in a 96 well block (Thomson Instrument Company, Oceanside, CA) at 37°C in 1 ml of LB media supplemented with 30 µg/ml kanamycin, and 34 µg/ml chloramphenicol for E. coli Rosetta (DE3), using a Shel Lab SI6R-HS shaking incubator with shaking at 650 rpm. The following day a 96 well block with 1 ml of fresh media supplemented with the appropriate antibiotics was inoculated with 50 µl of the overnight culture. The cultures were grown to an OD600 of 0.5 and protein expression induced by the addition of IPTG to a final concentration of 0.5 mM. The cultures were grown for an additional 4 hours at 37°C or, alternatively, were grown overnight at 18°C. Cultures were harvested by centrifugation and cell pellets were frozen and stored at −20°C pending lysis. The cell pellets were thawed and were resuspended in 500 µl lysis buffer A (20 mM HEPES, pH 7.5, 50 mM sucrose, 1 mM EDTA, 10 mM β-mercaptoethanol) supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO), 1 mM PMSF, and ∼40 U/mL Ready-Lyse lysozyme (Epicentre, Madison, WI). The cell suspensions were shaken at 800 rpm for 20 minutes at 25°C and then MgCl2 was added to 5 mM and DNase I to 20 µg/ml. At this point 500 µl of lysis buffers B (50 mM HEPES, pH 7.8, 0.3 M NaCl), C (50 mM HEPES, pH 7.5, 1 M NaCl), or D (50 mM HEPES, pH 7.5, 0.3 M NaCl, 0.2% LDAO) were added to the cell lysate and the suspensions shaken for an additional 20 minutes. The lysates were clarified by centrifugation and the supernatants were transferred to a fresh block containing 50 µl of Ni-NTA agarose beads (Qiagen, Valencia, CA) and were incubated with shaking for one to two hours at 4°C. After incubation the suspensions were transferred to a 96-well filter plate and the beads were washed three times with 1 ml of wash buffer B (50 mM HEPES, pH 7.8, 0.3 M NaCl, 10 mM imidazole), C (50 mM pH 7.5, 1 M NaCl, 10 mM imidazole), or D (50 mM HEPES, pH 7.5, 0.3 M NaCl, 0.2% LDAO, 10 mM imidazole) prior to elution of the bound complexes by the addition of 100 µl of elution buffers B (50 mM HEPES, pH 7.8, 0.3 M NaCl, 0.3 M imidazole), C (50 mM pH 7.5, 1 M NaCl, 0.3 M imidazole), or D (50 mM HEPES, pH 7.5, 0.3 M NaCl, 0.2% LDAO, 0.3 M imidazole). Eluates were analyzed by SDS-PAGE using Criterion gels (Bio-Rad Laboratories, Hercules, CA).
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