NMR sample preparation

Labelled ammonium chloride (15_NH

4Cl) and glucose (13C-D-glucose) were obtained from

15_{N labelled 3C}

15_{N-labelled 3C was prepared in a similar manner as described above. Typical preparation}

procedures are described. Competent BL21cod+ _{E. coli were transformed with H-MBP-3C}pro

using the heat-shock method. One colony of the plasmid-containing bacteria was inoculated into 600 ml of LB containing ampicillin at 100μg· ml−1and chloramphenicol at 34μg ·ml−1

(LBamp+/cam+_{). The culture was grown overnight at 30 °C (12hrs). The overnight culture was}

spun down at 1541 ×g (Sorvall GS3) for 15 mins and re-suspended in 300 ml of M9 minimal medium containing 100μg·ml−1ampicillin and 34μg·ml−1chloramphenicol (M9amp+/cam).

The culture was agitated for an hour at 30 °C. 3 g of glucose and 1 g of15_NH

4Cl were added via

sterile filter. After 30 minutes the cells were induced by the addition of 300μL 0.5 M IPTG for

a final concentration of 0.5 mM. When the stationary growth phase was achieved, the cells were harvested by spinning down at 1541×g(Sorvall GS3) for 15 mins. The final OD600was between

4 and 7. The resulting cell pellets were re-suspended in 25 ml of lysis buffer containing one Com- plete™ EDTA-free protease inhibitor tablet (Roche) and placed on ice. The resulting slurry was passed twice through a French press at 6,500 psi. The resulting lysed cells were centrifuged at 17,590×g(Sorvall SS34) for 30 minutes. The supernatant from this process was decanted from the pellet and sterile filtered with an 0.8μm filter. The filtered solution was then added to 3.5 ml

(bed volume) of Ni2+_{-NTA affinity medium and nutated overnight at 4 °C. The overnight slurry}

was poured into a Poly-Prep©_{column (Bio-Rad, CA) and the flow-through collected. The column}

was subsequently washed with 8 ml of lysis buffer. The column was then successively washed with 8 ml aliquots of lysis buffer containing 20 mM, 40 mM and 80 mM imidazole respectively. The column was eluted with 8 ml lysis buffer containing 250 mM imidazole. 50 units of bovine thrombin (Sigma) was added to the eluate and this was transferred into a 3 kDa MWCO dialysis bag and left for 24 hrs at 4 °C dialysing into 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM DTT (Initial work used dialysis into 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 0.25 mM TCEP at this stage. Little cleavage was observed, however. The 50 mM–80 mM fractions were pooled and added to the sample, a further 50 units of thrombin were added and this was then dialysed for 24 hrs into 50 mM Tris-HCl pH 7.4, 300 mM NaCl as previously but with 1 mM DTT as a reducing agent instead of TCEP. This resulted in better cleavage and DTT was used as a reducing agent for later preparations). The sample was passed through a 3.5 ml Ni2+_{-NTA column and the subjected}

to two 5 ml washes of lysis buffer. The flow-through and the lysis buffer wash were pooled and dialysed into 20 mM Tris-HCl pH 7.5, 1 mM DTT and then run through a Q-sepharose column.

The flow through from this column was then concentrated to 525μl. 25μl of D2O (5%) was added

to produce the NMR sample. Final sample concentration was between 0.3 and 1.3 mM. The nmr tubes were flushed with argon and sealed to prevent oxidation. A representative gel is shown as Figure 2.1. The final 3C sample is pure within the limits of detection by Coomassie staining.

21

31

a b

d

MBP

3C

c

Figure 2.1:SDS-PAGE gel of samples from final 3C purification steps. Lane (a) contains the fusion pro- tein post thrombin cleavage (b) contains 3C post 2nd pass through the Ni2+_{-NTA column (c) contains} 3C after final Q-sepharose polishing and (d) contains molecular weight standards.

13_C-15_{N-labelled 3C}

Typically, a flask containing 600ml of LBamp+/cam+_{was inoculated with 1 colony of H-MBP-}

3Cpro_{. This was left overnight shaking at 37 °C. The overnight culture was spun down at 1541×g}

and the cells resuspended in 300 ml of M9amp+_{giving an OD}

600of 3.0. The culture was left for 30

mins and 3 g of13_{C-D-glucose and 1 g of}15_NH

4Cl were added to the culture via sterile filter. After

1 hour the expression was induced by addition of 0.072 g of IPTG giving a final concentration of 1 mM. The cells were harvested when the growth curve flattened out (final OD600was∼6.5) by

spinning down at 1541×gfor 15 mins at 4 °C. The pellets were resuspended in 30 ml of lysis buffer containing one Complete™ EDTA-free protease inhibitor tablet (Roche). The resuspended cells were passed either through a cell disruptor three times at 13,000 psi or a French press three times at 6,000 psi. The resulting lysed cells were centrifuged at 17,590×g(Sorvall SS34) for 30

minutes. The supernatant from this process was decanted from the pellet and sterile filtered with an 0.8μm filter. The filtered sample was combined with 2 ml (bed volume) of Ni2+-NTA column

pre-equilibrated with lysis buffer and left nutating overnight at 4 °C (it was found that efficient binding to Ni2+_{-NTA could be achieved by passing the filtered sample under gravity through}

the resin in column format, this was much quicker and thus was used for later preparations). The slurry was then placed into a Poly-Prep© _{column (Bio-Rad, CA) and washed sequentially}

with two 4 ml aliquots of lysis buffer. Two further 5 ml washes of lysis buffer containing 50 mM imidazole and 100 mM imidazole were carried out. Finally the protein was eluted with 5 ml of lysis buffer containing 250 mM imidazole. All three imidazole-containing fractions were found to contain fusion protein so they were pooled (further preparations used only one 250 mM imidazole elution after the imidazole-free lysis buffer washes). 50 units of thrombin (Sigma) were added and the sample was dialysed into 2 l of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM DTT for 72 hrs. The resulting sample was passed through a Ni2+_{-NTA column pre-equilibrated}

with lysis buffer. The sample was then dialysed into 2 l of 20mM Tris-HCl pH 7.5, 1 mM DTT overnight and then passed through a 3.5 ml Q-sepharose column pre-equilibrated with 20 mM Tris-HCl pH 7.5, 1 mM DTT. Purity was increased in later work by raising the pH of this buffer to 7.8, this increased the affinity of protein contaminants for the Q-sepharose column. The sample was concentrated to 550μL and buffer exchanged into 20 mM potassium phosphate pH 6.5, 15

mM DTT, 0.5 mM EDTA. Later preparations used 50 mM BTM (50 mM bis-Tris propane / 50 mM MES (2-(N-morpholino)ethanesulphonic acid)) pH 6.0, 15 mM DTT 0.5 mM EDTA in the NMR sample buffer. 5% D2O was added and the sample was placed into a standard 5 mm NMR tube.

Yields of pure 3C were between 13 and 30 mg per litre of cell culture. The typical final protein concentration in NMR samples was between 11–22 mg·ml−1_{(0.5–1 mM).}