Protein expression and purification.
Human IκBα (67-287) was expressed in the Pet 11a vector and purified as previously described 14
. Two mutations were introduced into the p65 gene, N-terminal cysteine and a Cys38 to Ser to allow specific biotinylation only at the N-terminus. This gene will be referred to as p65. Murine NF-κB(p50248-376
) was co expressed in a modified pET 29b vector and purified as previously described 35
. Cells were harvested and sonicated then centrifuged at 12,000 RPM for 45 min and the supernatant loaded onto a tandem fast flow Q and fast flow S column (GE Healthcare) equilibrated in 50 mM NaCl, 25 mM Tris, pH 7.5, 0.5 mM EDTA. After loading the Q column was disconnected and protein fractions eluted from the S with a gradient from 50 to 400 mM NaCl. Fractions were collected and analyzed by SDS PAGE. Bands were visualized by silver staining and fractions with equal intensity of P50 and p65 were collected and pooled. The final step of the purification was size exclusion on an S-200 Superdex column equilibrated in 150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide. The purified fractions were biotinylated by incubation with a 1:1 molar ratio of biotin PEO maleimide (Pierce Chemicals), at room temperature for 30 min and purified immediately by size exclusion chromatography on an S200 Superdex 16/60 column. Fractions containing the biotinylated heterodimer were collected and stored at -80 °C in 50 μl portions until used.
All other NF-κB constructs were expressed using a Pet 11a single expression vector and purified using a similar tandem column technique. Again, an N-terminal cysteine was introduced into the p65 as described above. For the NF-κB p65190-321 and NF-κB p50248-350 constructs, E. coli BL21 DE3 cells were grown to an OD of 0.6 and induced at room temperature for 16 hours with 0.1mM IPTG. For the NF-κB p65190-321 the column was equilibrated in 50mM NaCl, 25 mM MES pH 7.0, 10 mM BME, 0.5 mM EDTA. For the NF-κB p50248-350 the column was equilibrated in 50 mM NaCl, 25 mM MES pH 6.2, 10 mM BME, 0.5 mM EDTA and gradient of 50-300 mM NaCl, was run.
For the NF-κB p651-325, NF-κB p651-304 and NF-κB p5039-363 proteins, cells were induced with 0.5 mM IPTG. NF-κB p651-325 the columns were equilibrated in 50 mM NaCl, 25 mM MES, pH 6.5, 10 mM BME, 0.5 mM EDTA, a gradient was run from 50-450 mM NaCl. For NF-κB p651-304 50 mM NaCl, 25 mM Tris, pH 7.0, 10 mM BME, 0.5 mM EDTA and a 50-300 mM gradient was used. For NF-κB p5039-363 the columns were equilibrated in 50 mM NaCl, 25 mM MES, pH 6.2, 10 mM BME, 0.5 mM EDTA. Gradient was run from 50-700 mM NaCl.
Protein concentrations were determined spectrophotometrically from a scan of wavelengths 340-220 nm using the following ε280 values: 24180 for NF-κB p50248-350, 21620 for NF-κB p65190-321, 19060 for NF-κB p65190-304, 36980 for the NF-κB p651-325, 34420 for NF-κB p651-325, and 42100 for NF-κB p5039-363 homodimers and 30580 for NF-κB(p50248-376/p6519-325), 22900 for NF-κB(p50248-350/p65190-321), 21620 for NF-κB(p50248-350/p65190-304), 39540 for NF-κB(p5039-363/p651-325), 38260 for NF-κB(p5039-363/p651-304) heterodimers. For IκBα67-287 an ε280 of 12090 was used. Dimers were formed in vitro by incubating an equimolar amount for two hours at 25 °C and overnight at 4 °C prior to ITC experiments. For SPR experiments, the p65 was biotinylated as already described. A 1000 fold excess of un-biotinylated p50 (for heterodimers) or p65 (for homodimers) was incubated with biotinylated p65 and the equilibrated mixture was immobilized immediately on a streptavidin (SA) SPR chip.
The C-terminal residues 289-320 of NF-κB were expressed in the trp leader vector which contains an octa-histidine tag and thrombin cleavage sequence and drives small peptides into inclusion bodies 36
. Inclusion bodies were solubilized 6M guanidine hydrochloride, 50mM Tris, pH 7.4 and the solubilized peptide was captured by Ni-NTA column equilibrated in the same buffer, and a gradient was run to a final concentration of 150 mM NaCl, 50 mM Tris, pH 7.4, 2mM CaCl2
. The peptide was cleaved from the column with thrombin on the column for 4 hrs at 25 °C. The final purification step was reverse phase HPLC on a C18 column with a 0-50% acaetonitrile gradient, with 0.1% TFA. The peptide was lyophilized and dissolved in 150mM NaCl, 10mM MOPS, pH 7.5, 0.5mM EDTA and the pH adjusted with 10 M NaOH.
Sensorgrams were recorded on a Biacore 3000 instrument using streptavidin (SA) chips. Biotinylated NF-κB was immobilized on the chip in a high salt buffer (500 mM NaCl, 10mM Tris, pH 7.5, 0.5mM EDTA, 0.5mM sodium azide, 0.005% P20). Sensorgrams were run in the automatic subtraction mode using flow cell 1 (FC 1) as an ummodified reference. Data was collected for FC's 2,3 and 4, which contained varying amounts of NF-κB ligand with the lowest amount immobilized on FC2 and the highest on FC4. Injections were made using the kinject injection mode, alternating highest with lowest concentration samples, with a 5 minute contact time and a1200 second dissociation phase, in all cases except for the weaker interactions where a 3 min contact time and a 3 min dissociation phase was used. The running buffer used for the binding experiments was 150 mM NaCl, 10mM Tris, pH 7.5, 10% (w/v) glycerol, 3mM DTT, 0.5mM sodium azide, 0.2 mM EDTA and 0.005% P20. The glycerol improved the stability of the NF-κB during regeneration. Regeneration was achieved using a one min pulse of a urea solution. The concentration of urea required depended on the NF-κB construct and the experimental temperature and was prepared by diluting a 6M stock into the running buffer. The minimum urea concentration required for complete regeneration under each condition was determined by repeat injections. The data was analyzed using the Bia Evaluation 4.1 software using a simple 1:1 langmuir binding model. Between 3 and 12 sensorgrams were obtained for each construct and condition tested using a range of immobilized NF-κB and IκBα concentrations.
at 37 °C, 200, 300 and 400 RU of NF-κB were immobilized. 0.23 to 4.0 nM IκBα was injected. A one min pulse of 3M urea was used for regeneration. Lower ligand and analyte concentrations were not employed to avoid the long term noise of the instrument becoming significant relative to the slow dissociation of the IκBα 37
For NF-κB(p50248-350/p65190-321 sensorgrams were obtained at 25 and 37 °C. NF-κB was immobilized at 50, 75, 100, 150, 200, 250 and 350 RUs. Sensorgrams were recorded using several ranges of IκBα. These were: 0.87 to 9.9 nM IκBα with 200, 250 and 350 RU NF-κB, 0.24 to 20 nM IκBα for 50, 75 and 100 RU of NF-κB and with 0.01 to 5 nM IκBα with 100, 150 and 200 RU of NF-κB at 37 °C. For experiments at 25 °C, concentrations used were 0.87 to 9.9 nM IκBα with 100, 200 and 300 RU NF-κB, 0.24 to 20 and 0.022 to 10 nM IκBα with 50, 75 and 100 RU NF-κB. A one min pulse of 1.5 M urea was used for regeneration at 37 °C and 3 M Urea at 25 °C.
For NF-κB(p50248-350/p6519-304 at 25 °C, 16 to 1000 nM IκBα was used with 100, 200 and 250 RU of NF-κB, and 9.9 to 5000 nM IκBα was used with 50, 75 and 100 RU of NF-κB. At 15 °C, 9.9 to 5000 nM IκBα was used with 50, 75 and 100 RU of NF-κB. No regeneration was required at either temperature with this NF-κB construct because it bound so weakly. Data were analyzed by equilibrium analysis in addition to the kinetic analysis. The equilibrium response was plotted against the IκBα concentration and a line was fit to R = KA × [IκBα].Rmax/(KA.[IκBα]+1) where R is the equilibrium response at a specific IκBα concentration, Rmax is the response at saturation of the ligand on the chip and KA = 1/KD.
For the NF-κB(p50248-350/p6519-304), the effect of the 10% glycerol in the running buffer was assessed by experiments at 25 °C with 9.9 to 5000 nM IκBα and 50, 75 and 100 RU of NF-κB immobilized using a running buffer that was the same as that used for the ITC experiments (150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide, 0.005% p20). No significant difference in the binding data was observed using this buffer.
ITC experiments were carried out on a Microcal MCS instrument. IκBα and NF-κB were purified by size exclusion chromatography on an S-75 or S-200 column respectively immediately prior to use. In a typical ITC experiment, 20 15 μl injections of 50 μM NF-κB were made into a 5 μM IκBα solution in the cell. ITC experiment were carried out in a buffer of 150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide. Isotherms were analyzed using the Origin software (Microcal) as described elsewhere 38
. For the very tight complexes, the KD,obs
could not be determined due the high `c' value for the interaction, where c is defined by Wiseman et al. 38
Surface area calculations