| Chen FE, Kempiak S, Huang DB, Phelps C, Ghosh G.
(1999)
Protein Engineering,
12,
423-428 |
| Functional Studies |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 25.0 |
| overnight |
| pET29b |
| Refolding of the p50/p65 heterodimer
Truncated recombinant murine p50 RHRL (residues 39–364)
and p65 RHRs (residues 19–291) proteins were overexpressed
separately in E.coli cells with T7 promoter driven expression
plasmids (Novagen). The proteins were purified independently.
Cells overexpressing p50 protein were lysed by sonication
Purification and functional analysis of p50/p65 NFkB
in 20 mM NaCl, 10 mM MES pH 6.0, 10 mM bME, 0.5 mM
EDTA and 0.1 mM PMSF and the cell debris removed by
centrifugation in a SS-34 rotor (Sorvall) at 12 000 r.p.m.
(11 000 g) and 4°C for 30 min. A polyamine P precipitation
was performed to a final concentration of 1% and the insoluble
fraction removed by centrifugation in a SS-34 rotor (Sorvall)
at 12 000 r.p.m. (11 000 g) and 4°C for 20 min. The cleared
lysate was diluted with more lysis buffer and passed over a
gravity SP-Sepharose Fast Flow cation exchange column
(Pharmacia). The column was washed with lysis buffer until
A280 was less than 0.1 and the protein eluted with a linear
gradient of 20–500 mM NaCl in lysis buffer at 4°C. The peak
protein fractions were loaded onto a Mono S cation exchange
FPLC column (Pharmacia) and eluted with a linear gradient
of 50–400 mM NaCl in 10 mM MES pH 6.0 and 5 mM bME
at room temperature.
Cells overexpressing p65 protein were lysed by sonication
and cleared by centrifugation. A streptomycin sulfate precipitation
was performed to a final concentration of 0.5%. The
insoluble fraction was removed by centrifugation in a SS-34
rotor (Sorvall) at 14 000 r.p.m. (15 000 g) and 4°C for 30 min,
and the cleared lysate dialyzed against lysis buffer. The protein
425
was then loaded onto a gravity SP-Sepharose Fast Flow cation
exchange column (Pharmacia) and washed with lysis buffer
until A280 , 0.1. The protein was eluted with a linear gradient
of 20–500 mM NaCl in lysis buffer. Peak fractions were
pooled. We have no reason to believe that the different
purification protocols for each protein are not interchangeable.
P50 and p65 proteins were unfolded with a slight molar |
| IPTG |
| OD 0.4 =
600 |
| Sonication |
| None |
| Ion-exchange chromatography |
| soluble |
| Dialysis |
| unknown |
| 7 M Urea, 0.5 M NaCl, 1 mM EDTA, 0.2 mM PMSF, 10 mM BME, 25 mM Tris HCl (pH 7.5) |
| 0.5 M NaCl, 1 mM EDTA, 0.2 mM PMSF, 10 mM BME, 25 mM Tris HCl (pH 7.5) |
| Ion-exchange chromatography |
| no tag |
| 7.5 |
| 4.0 |
| n/a |
| n/a |
| Beta-mercaptoethanol |
| n/a,n/a,n/a,n/a |
| P50 and p65 proteins were unfolded with a slight molar excess of p65 and a final total protein concentration of 154– 666 mg/ml in 7 M urea, 0.5 M NaCl, 1.0 mM EDTA, 0.2 mM PMSF, 10 mM bME and 25 mM Tris–HCl, pH 7.5. The proteins were then refolded by dialyzing in 8000 MWCO tubing (Spectrum) for 6 h at 4°C in 2 liters of the same buffer without urea. The buffer was changed two more times and then exchanged for a final time with 2 liters of 25 mM Tris– HCl pH 7.5 and 20 mM NaCl for a total of four dialysis events. The lysate was then loaded onto a cation exchange SPSepharose fast flow column and the protein purified with a linear salt gradient at 20–300 mM NaCl in 25 mM Tris–HCl, pH 7.5. Pooled peak fractions were concentrated, aliquoted and stored at 280°C. The refolding procedure yielded 15 mg of clean heterodimer from approximately 50 mg of each protein initially used. |
| DNA binding |
| None |
| None |
| n/a |
| 15 mg (30%) |
| n/a |
| n/a |