Protein expression and purification

2.3.1 Transformation

To transform the plasmid into bacterial cells, 1 µl of pET28-Hsp90-NTD plasmid was added to a 50 µl aliquot of competent E.coli BL21 (DE3) cells. The tube containing the DNA and the bacterial cells was left to incubate on ice for 30 minutes. Following the incubation, the tube was placed on a 42°C heat block for 45 seconds, after which 500 µl of LB broth was added and the cells were placed in a shaker (250 rpm) to grow for 1 hour at 37°C. The cells

were centrifuged briefly and the cell pellet re-suspended to 100 µl of LB broth before plating them onto LB-Agar plates containing kanamycin antibiotic for selecting the plasmid containing cells.

2.3.2 Preparation of DNA stock

To prepare plasmid stock for Hsp90-NTD expression, the pET28a-6His-TEV- Hsp90(D9-E246) was transfected into E.coli DH5α cells following the heat shock method described above. A colony from the plate was picked to grow in 5 ml LB plus kanamycin overnight at 30°C. The following morning, the cells were harvested by centrifugation and DNA extracted using the Invitrogen Purelink Quick plasmid miniprep kit for plasmid DNA extraction according to manufacturer’s instructions.

2.3.3 Protein expression trials

To optimise the expression protocol for the Hsp90-NTD using the M9 media, a small scale protein expression trial was carried out. For the trial, the effects of different expression temperatures (37°C, 30°C and 20°C) and IPTG amounts (0.1 mM and 1 mM) were tested.

First, a transformed E.coli BL21 (DE3) colony containing the pET28a-Hsp90- NTD plasmid was picked from the LB-Agar plates and added into 5 ml of LB/kanamycin media. The bacteria were left to grow in an incubator for 4 hours (37°C, 250 rpm). After this, 1 ml of the starter culture was taken to seed 25 ml cultures. The cells were further grown as above, until the, optical density of the culture measured at 600 nm (OD600) was around 1.00. After this, the expression of the plasmid encoded Hsp90-NTD was induced with either 0.1 mM or 1 mM IPTG and the culture was left to grow at either at 37°C for 4 hours or at 30°C or at 20°C overnight. Samples from the cultures were taken before and after the induction and these were run in SDS-PAGE gel, as specified in Section 2.2.3.

2.3.4 Large scale protein expression (un-labelled)

To express the Hsp90-NTD protein, a transformed E.coli BL21 (DE3) colony containing the pET28a-Hsp90-NTD plasmid was picked from the LB-agar plates and added into 5 ml of LB/kanamycin media. The bacteria were left to

grow in an incubator for four hours (37°C, 250 rpm). After this, about 2 ml of the starter culture was taken to seed 50 ml of M9 media in a baffled flask. The cells were further grown as above until the optical density of the culture measured at 600 nm (OD600) was around 1.00. Next, the culture was transferred to 500 ml M9 media to grow further until the OD600again reached 1.00. After this, protein expression was induced by addition of 1 mM IPTG to a final concentration and the culture was left to grow overnight at 20°C. The following day, the cells were harvested by centrifugation (5000 rpm for 20 min at 4°C). After this the samples were kept on ice to prevent any proteolysis and the pellet was re-suspended in 35 ml of Buffer A (40 mM HEPES pH 8, 300 mM NaCl and 5 mM DTT). The pellet was then flash frozen at -80°C until purification.

2.3.5 Expression of labelled protein

The above protocol was used to produce labelled protein, except that the starter culture was grown overnight at 30°C to allow for the slower bacterial growth in deuterium oxide.

2.3.6 Protein purification

To purify the His-tagged Hsp90-NTD, the cell pellet was defrosted. After this 20 mg/ml lysozyme and a protease inhibitor P8849 tablet (Roche) were added to the sample. The tube was briefly mixed, and the cells were left to incubate on ice for thirty minutes. After incubation, the sample was sonicated using a six seconds on, six seconds off cycle for ten cycles, after which the lysed cells were then centrifuged for 20 minutes at 20,000 rpm.

After centrifugation, the soluble fraction containing the His-tagged Hsp90-NTD was loaded onto a HisTrap HP column (GE Health Care) using the ÄKTA FPLC purification system. The unbound fraction was collected after which the column was washed first with five column volumes of Buffer A containing 10 mM imidazole followed by a second wash again with five column volumes of Buffer A containing 20 mM imidazole. Finally, the His-tagged protein was eluted with Buffer A containing 250 mM imidazole and the fractions containing the Hsp90-NTD were collected.

2.3.7 Buffer exchange

Following the purification, the fractions containing the purified Hsp90-NTD were pooled and dialysed against 500 ml of 20 mM sodium phosphate, pH 7.5 buffer (20 mM Na2HPO4and 20 mM NaH2PO4) (NaPi buffer) for around twenty four hours at 4°C with two changes of the dialysis buffer.

If the protein sample required concentrating, this was done using a 10 K Amicon centrifuge tube until the desired concentration (350 μM for NMR samples) was reached.

2.3.8 NMR sample preparation

The samples for NMR measurements contained additionally 1% AEBSF hydrochloride protease inhibitor (from Biochemica) and 5% D2O to allow the lock signal setting that stabilizes the magnetic field strength of the NMR instrument.

2.4 Isothermal titration calorimetry

The isothermal titration calorimetry (ITC) experiments were performed using a MicroCal iTC-200 instrument at 25°C. For the measurements, 20 µM Hsp90- NTD and 200 µM GVK0153 ligand, 400 µM 17-DMAG or 300 µM of CNF2024, geldanamycin, GVK1061 or radicicol were prepared in 20 mM sodium phosphate buffer (20 mM Na2HPO4, 20 mM NaH2PO4, pH 7.5) assay buffer with matched 2% DMSO concentration in both the protein and ligand samples. For the experiments, 200 µl of 20 µM Hsp90-NTD protein solution was placed in the ITC instrument sample cell and the concentrated ligand solution in the 40 µl syringe. Nineteen injections of 2 µl of ligands were added into the protein sample and the mixing/equilibration time between samples injections was set to 150 seconds.

The ITC data were processed using an automated algorithm in Nitpic software to correct the base line [108]. The titration data was fitted using one site model and a non-linear least squares curve-fitting Levenberg and Marquardt algorithm with the Origin 7 Software from MicroCal.