Materials and Methods
2.4.3 Determination of recombinant protein expression by pilot-scale affinity purification
Isolation of the total soluble E. coli cellular protein fraction
Frozen cell pellets were resuspended in sonication buffer, at 5ml/g of cell pellet. Sonication buffer is composed of 50mM Tris-base (pH 7.2 at 21°C), 300mM KCl, 20mM imidazole, 0.01% Triton™ X-100 (Sigma-Aldrich®), and 10mM β-mercaptoethanol.
50µl of Protease Inhibitor Cocktail for use in purification of Histidine-tagged proteins (protease inhibitor; Sigma-Aldrich®) and either 0.5 or 1 mg/ml of Lysozyme from chicken egg white (lysozyme; Sigma-Aldrich®) was added to the resuspended cell solution. The resuspended cell solution was then incubated on ice for an hour, and then the cells were sheared through physical disruption by sonication. Sonication was performed with an Ultrasonics Inc. Cell Disruptor Model W-225R for 6 rounds of 10 seconds, with 20 second refractory period, on ice at power level four. The cell lysate was then centrifuged at 15000 RCF for 30 minutes at 4°C. The cleared cell lysate was then siphoned from the cell debris pellet, and kept on ice until purification, or a stored at -20°C.
Isolation of insoluble E. coli cellular protein fraction
After the soluble protein was removed from the lysed cell pellet, the resultant pellet would be resuspended in sonication buffer containing 8M urea, through sonication. The insoluble protein would then be then centrifuged at 15000 RCF for 30 minutes at room temperature, and the supernatant collected and kept on ice until purification, or a stored at -20°C.
32 Pilot-scale glutathione affinity purification
Pilot-scale purification of GST-tagged proteins was performed making use of glutathione- affinity purification from the cleared cell lysate using glutathione-agarose (Sigma- Aldrich®). In brief, 50μl of 40% (v/v) glutathione-agarose resin slurry in 2M NaCl and 1mM sodium azide, was equilibrated in 1ml of sonication buffer containing 0.1% Nonidet™ P 40 Substitute (NP 40; Sigma-Aldrich®). The resin was settled by brief centrifugation at 350 RCF for 1 minute, and the sonication buffer siphoned from the settled resin. 1ml of cleared cell lysate with 0.1% NP 40 was added to the resin and then mixed for 1 hour at 4°C. The glutathione-agarose was then recovered by brief centrifugation, and the unbound protein fraction siphoned off and stored on ice. The resin was washed once in 1ml of sonication buffer, and moved to a sterile microcentrifuge tube. The resin would then be suspended in 2×SDS loading dye (typically 45µl in volume) and the proteins heat denatured at 95°C for 5 minutes. The resultant denatured protein would then be moved to a fresh tube for SDS-PAGE analysis, or a stored at -20°C.
1) Pilot-scale nickel affinity purification
Pilot-scale purification of the hexa-histidine (6His)-tagged proteins was performed making use of nickel-affinity purification from the cleared cell lysate using PureProteome™ Nickel Magnetic Beads (Merck Millipore; Ni-beads). In brief, 5 or 10µl of suspended beads would be pipetted into a sterile microcentrifuge tube. The beads were separated from the storage buffer using a magnetic separator stand. The beads were equilibrated in 1ml of sonication buffer (with or without 0.1% NP 40), and recovered using the magnetic separator. 1ml of cleared cell lysate was added to the beads, and mixed for 1 hour at 4°C. The nickel magnetic beads were recovered with the magnetic separator, and the unbound protein fraction removed and stored on ice. The beads were washed either 3 times in 50µl of sonication buffer, or once in 1ml of sonication buffer. The resuspended beads were moved to a sterile microcentrifuge
33 tube. The beads were suspended in 2×SDS loading dye (typically 30µl in volume) and heated at 95°C for 5 minutes to denature the proteins. The resultant denatured protein would then be moved to a fresh tube for SDS-PAGE analysis, or a stored at -20°C.
SDS-PAGE analysis
SDS-PAGE gels were either poured in custom SDS-PAGE casting plates, and electrophoresis performed with custom gel apparatus, or the Bio-Rad mini-gel system was used. The volumes of stacking and running gel were adjusted accordingly. Typically the custom gels required 5ml of the 5% stacking gel and 10ml of the 12 resolving gel. The Bio- Rad mini-gels required approximately 1.25ml 5% stacking gel and 4.5ml 12% resolving gel.
Combs of 1mm width were used for all the gels.
Table 3: SDS-Page solutions
12% Resolving gel: Reagent Volume
Water 4.2ml
40% 37.5:1 bis-acrylamide/acrylamide mix (Sigma-Aldrich®) 3ml
1.5 M Tris-HCl pH 8.8 2.5ml
10% SDS solution 0.1 ml
Ammonium persulphate (100mg/ml) 0.2ml
Tetramethylethylenediamine (TEMED) 10µl
Total 10ml
5% Stacking gel: Reagent Volume
Water 3 ml
40% 37.5:1 bis-acrylamide/acrylamide mix (Sigma-Aldrich®) 0.625 ml
1.0 M Tris-HCl pH 6.8 1.25 ml
10% SDS solution 50µl
34
Ammonium persulphate (10% w/v in H2O) 0.1 ml
Tetramethylethylenediamine (TEMED) 5µl
Total 5ml
Samples were mixed to be in either 1 or 2×SDS loading dye (100 mM Tris-Cl (pH 6.8), 4%
(w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200mM dithiothreitol [DTT]) and heated to 90°C for 2 minutes. Samples were then briefly centrifuged to allow the condensate to mix into the sample. The samples were then loaded into the SDS-PAGE and electrophoresis begun, with the ampere kept constant at typically 35-45mA until completion.
The gels were removed from the glass plates, and washed in deionised water 3 times for 10 minutes, and then stained with Bio-Safe™ G-250 Coomassie premixed stain (Bio-Rad) for 30 minutes to an hour. The gels were de-stained by washing repeatedly with water. Gels were visually inspected to determine the presence of protein, and photographed.
2.4.4 Identification of 6His-PfTLP and 6His-PfTFIIB proteins by mass