The aims of this study were to purify and investigate the activity of Rv3915, as well as monitor the phosphorylation of Rv3915 and the influence of phosphorylation on the activity of this protein.
Bacterial Strains:
BL21 (DE3): Derived from B834 when transduced to Met+, an E. coli B strain with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq. Cells were grown aerobically in standard Luria-Bertani broth supplemented with kanamycin (50 µg/ml). Transformed plasmids containing T7 promoter driven expression are repressed until IPTG induction of T7 RNA polymerase from a lac promoter.
C41 (DE3): Derived from BL21 (DE3), an E. coli B strain with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq. Cells were grown aerobically in standard Luria-Bertani broth supplemented with kanamycin (50µg/ml).Transformed plasmids containing T7 promoter driven expression are repressed until IPTG induction of T7 RNA polymerase from a lac promoter.
C43 (DE3): Derived from C41 (DE3) through selection for resistance to a contrasting toxin protein, an E. coli B strain with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq. Cells were grown aerobically in standard Luria-Bertani broth supplemented with kanamycin (50µg/ml). Transformed plasmids containing T7 promoter driven expression are repressed until IPTG induction of T7 RNA polymerase from a lac promoter.
Competent Cells Preparation: 2ml LB bacterial starter culture was inoculated with a single colony of E.coli and incubated overnight at 37°C with shaking. 100ml LB bacterial main culture was inoculated with 1ml of starter culture and incubated at 37°C with shaking till OD580 ranged between 0.2 –0.4. Culture was placed on ice for 15mins, than centrifuged at 2500g and 4°C for 15mins. Supernatant was discarded and pellet was re-suspended in 50ml of pre-chilled 100mM CaCl2. Mixture was placed on ice for 5mins and then centrifuged again, discarded supernatant and gently re-suspended pellet in 50ml of 100mM CaCl2. Placed on ice for 15mins, and then repeated centrifugation. The supernatant was discarded and gently re-suspended pellet in 2ml of 100mM CaCl2 and 15% glycerol. A 100µl aliquots were made into pre-chilled eppendorf tubes and stored at -80°C.
E.coli Transformation: 100µl aliquot of competent cells was thawed and transferred into a pre-chilled 14ml falcon tube and 1µl of DNA (concentration between 20-200ng/µl) was added and left on ice for 15mins. A pre-warmed medium (900µl) was added to cells and incubated at 37°C with shaking for 1hr. For each transformation, 3 LA plates were used, containing an appropriate antibiotic: plated 100µl of the undiluted culture, 100µl from 10-1 dilution and 100µl from 10-2 dilution. The plates were Incubated overnight at 37°C. For the negative control, 10µl of competent cells was mixed with 100µl of the recovery medium and incubated at 37°C with shaking for 1hr.
Preparation of E.coli cultures for protein expression at 20°C: Four 5ml bacterial starter cultures, containing 5µl ampicillin or streptomycin, each inoculated with a single colony of E.coli and incubated at 37°C with shaking overnight. Four 500ml bacterial starter cultures, containing 500µl ampicillin or streptomycin, each inoculated with a 2ml of starter cultures and incubated at 37°C with shaking until A580 ranged between 0.6-0.85. Left on ice for 1hr, 5ml of IPTG added and cultures incubated overnight at 20°C with shaking. All main cultures were centrifuged together at 7000G and 4°C for 25mins. The supernatant was discarded, re-suspended pellet in 5ml of H20 and centrifuged at 14,000G and 4°C for 10mins after which the supernatant was discarded and placed pellets at -20°C.
Preparation of E.coli cultures for protein expression at 37°C: Four 5ml bacterial starter cultures, containing 5µl ampicillin (stock: 100µg/ml) or streptomycin (stock: 20µg/ml), each inoculated with a single colony of E.coli and incubated at 37°C with shaking overnight. Four 500ml bacterial starter cultures, containing 500µl ampicillin or streptomycin, each inoculated with a 2ml of starter cultures and incubated at 37°C with shaking until A580 ranged between 0.6-0.85. A 1 ml Pre-aliquot of cells and media was taken from each main culture and placed on ice in sterile Eppendorf tubes. 5ml IPTG added to remaining cells in main cultures. Main cultures were incubated for 4 hrs at 37°C with shaking. 1 ml Post-aliquot of cells and media was taken from each main culture for each hour and placed on ice in sterile Eppendorf tubes. All main cultures were centrifuged together at 7000G and 4°C for 25mins. The supernatant was discarded and re-suspended pellet in 5ml of water and centrifuged at 14,000G and 4°C for 10mins, discarded the supernatant and placed pellets at -20°C.
Purification of Rv3915 from iinclusion bodies: 500ml induction pellet were re-suspended in 40ml of lysis buffer (25mM Tris pH 8.0, 150mM NaCl, 0.5% Triton-X100, 1Mm EDTA) and sonicated using a macroprobe; 8-10 pulses, each pulse 30secs with a 1min break between each pulse. Incubated at room temperature with shaking for 30mins after which 5mM MgCl2 was added and incubated again at room temperature with shaking for 15mins, centrifuged at 20,000G and 4°C for 20mins. The supernatant was discarded and pellet re-suspended in 40ml of 25mM Tris pH 8.0, 0.5M NaCl, 0.5% Triton-X100 and 1mM EDTA, centrifuged, discarded supernatant and the pellet re-suspended in 40ml of 25mM Tris pH 8.0, 0.5M NaCl and 1M urea. The sample was centrifuged, after which the supernatant was discarded and re-suspended pellet in 25mM Tris pH 8.0. 100µl aliquots of sample was made into eppendorf tubes, centrifuged and stored at -80°C.
Affinity Chromatography: After removal of storage solution, the column was washed once with urea, rinsed once with EDTA, washed with MilliQ three times, nickel loading solution added once to column and rinsed column three times with buffer A (5ml of 5M Nacl, 25ml of 1M Tris, made up to 1 litre). 500ml bacterial induction pellet was thawed and re-suspended in 10ml of buffer B (200ml of 5M NaCl, 25ml of 1M Tris, made up to 1 litre), sonicated using a macroprobe; 6 pulses, each pulse 40 seconds with a 40 seconds break between each pulse. The sample was aliquoted into 2ml eppendorf tubes and centrifuged at 15,000g and 4°C for 30mins. The supernatants were pooled and the pellets discarded. The nickel column was rinsed once with buffer B, then loaded pooled supernatant sample. The column was washed twice with 60mM imidazole and eluted 1ml fractions with 400mM imidazole.
Gel Filtration: Form analytical gel filtration analysis Hiload 16/600 Superdex 200pg column was used. Equilibrated column with PBS buffer. Protein (1ml), purified on Ni-sepharose at a concentration of 1mg/ml, was filtered using 0.2 micron filter and injected to the column at a flow rate of 1ml/min. The chromatography was carried on protein AKTA system equipped with multi-wavelength UV/Vis detector set at 260 and 280nm, at flow rate of 1ml/min.
Dephosphorylation Assay: Purified culture supernatant on DEAE-sepharose ion exchange column in lysis buffer (50mM Tris HCl pH 8.5, 50mM NaCl, 10mM KCl, 25mM EDTA, 10% Glycerol) and eluted in 25mM Tris pH 8.5, 1M NaCl and 10mM KCl. Purified eluted sample on Ni-sepharose column in 25mM Tris pH 8.5, 1M NaCl and 10mM KCl and eluted in 200mM imidazole. Pooled together and concentrated fractions with the highest amount of protein, incubated at room temperature or 37°C with CIP in CIP buffer (50mM Tris HCl pH 7.9, 100mM NaCl, 10mM MgCl2, and 1mM DTT) for 0, 0.5, 1 and 2hrs before the addition of 4xSRB.
SDS-PAGE: Samples were loaded and ran gel at 200V for 1hr. The gel was stained with Coomassie blue overnight and washed with water.
Zymogram Assay: 0.06g of Micrococcus luteus was added to 12% resolving gel mixture, in the absence of APS and TEMED. 60µl 10% APS and 20µl TEMED were added to the mixture and pipetted to fill ¾ of gel cassette and left to set for 30mins, with a layer of isopropanol as a cover. Standard 5% stacking gel was prepared and pre-ran zymogram at 200v for 30mins. Samples were loaded and ran gel at 200V for 1hr. The zymograms were washed for 20mins, three times, in PBS + 0.1% Triton solution, incubated overnight at 37°C in PBS + 0.1% Triton solution and stained with methylene blue for 1hr and rinsed with water.
Western Blotting: Blot was placed in 50ml falcon tube, added 30ml of PBS + 1% milk and washed for 20mins. The PBS solution was discarded and the primary antibody (1µl of anti-poly His/anti-phosphothreonine in 2ml PBS + 1% milk solution) was added to the blot, left blot rolling overnight at -20°C and washed for 10mins, three times, with 30ml of PBS + 0.1% Tween 20 solution. After which 5ml of secondary antibody was added (1µl of Anti-mouse IgG in 10ml PBS + 1% milk solution) and left blot rolling at RT for 2hrs, washed with PBS + 0.1% Tween 20 solution, placed membrane in flat horizontal container and added 2ml of BCIP®/NBT Liquid substrate solution. This was left in dark until the visualization of bands.