Department of Biology and 1 Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92037, USA
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Abstract |
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Keywords: NFB/protein analysis/protein crystallography/protein purification/recombinant expression
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Introduction |
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The preparation of particular recombinant dimers free of other dimer combinations is required for further in vitro NFB protein studies. Large scale expression of the p50/p65 heterodimer was needed not only to facilitate the crystallization and X-ray diffraction studies of the protein, but also to allow future biochemical studies of various deletion mutants. Electron density maps of Rel dimer protein RHR crystal structures have demonstrated that the C-terminal nuclear localization sequences (NLS) are not ordered (Ghosh et al., 1995
; Müller et al., 1995
; Chen,F.E. et al., 1998
). In light of this, various protein constructs with and without the NLS on each polypeptide were made and tested for crystallization (Cramer and Müller, 1997
).
We report two methods for the large-scale expression and purification of recombinant NFB p50/p65 heterodimer. The first utilizes a bacterial double expression vector which contains two ribosomal binding sites to facilitate the coexpression of the two polypeptides of the p50/p65 NF
B heterodimer. The second method uses a mixed protein refolding strategy. Both methods yield crystallizable protein that binds DNA with the same affinity in electrophoretic mobility shift assays.
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Materials and methods |
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A pET29b expression vector (Novagen) was manipulated to replace the thrombin site with another copy of the upstream ribosomal binding site between the KpnI and NcoI restriction endonuclease sites. The insert consisted of two annealed oligos synthesized via phosphoramidite synthesis on a Milligen Cyclone Plus DNA synthesizer. The two strands had the following sequence: 5'-CAAATAATTTTGTTTAACTTTAAGAAGGAGATATAC-3' and 5'-CATGGTATATCTCCTT-CTTAAAGTTAAACAAAATTATTTGGTAC-3' (ribosomal binding site boldface). A PCR cloned fragment of the truncated murine cDNA p50 RHRL (aa 39364) containing a C-terminal stop codon was inserted between the NcoI and SalI sites so that translation starts with the AUG of the NcoI site. After confirmation of IPTG inducible expression, a PCR fragment of the murine cDNA p65 RHRs (aa 19291) was inserted after the first ribosomal binding site between the NdeI and BglII sites so that translation starts with the AUG of NdeI. This construct was also tested for IPTG-inducible expression. A total of four constructs were made with and without the NLS on each protein: p50 RHRL/p65 RHRL, p50 RHRL/p65 RHRs, p50 RHRs/p65 RHRL and p50 RHRs/p65 RHRs. The p50 RHRS contains amino acids 39350, the p65 RHRL contains amino acids 19305, and both contain an extraneous N-terminal methionine residue from the AUG start sites.
A C-terminal expressed hexahistidine-tagged p50 RHRL/p65 RHRs protein was also constructed and yielded large amounts of protein, but was not pursued since it failed to yield diffraction quality crystals.
Purification of coexpressed protein
Two liters of Escherichia coli BL21[DE3] cells, grown in LuriaBertoli media at 37°C to an O.D.600 of 0.4, were induced with 0.1 mM IPTG and shaken overnight at room temperature. Cells were harvested by centrifugation in a SLA-3000 rotor (Sorvall) at 4000 r.p.m. (1 700 g) and 4°C for 20 min and were resuspended in a lysis buffer of 50 mM NaCl and 25 mM TrisHCl pH 7.5, 20 mM ßME, 0.5 mM EDTA and 0.1 mM PMSF. The cells were lysed by sonication on ice 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.
The cleared lysate was passed over a gravity Q-Sepharose Fast Flow anion exchange (Pharmacia) connected to a SP-Sepharose Fast Flow cation (Pharmacia) exchange column. Both columns were washed until the A280 was less than 0.1 and then the anion exchange column was removed. The protein was eluted with a linear gradient of 50400 mM NaCl in lysis buffer at 4°C. Peak fractions were identified at this and subsequent stages by Bradford assay and SDSPAGE and concentrated in a Centriprep-30 or Centricon-30 concentrator (Amicon). The peak protein fractions were desalted by dilution and loaded onto a Mono S cation exchange FPLC column (Pharmacia) and eluted with a linear gradient of 50200 mM NaCl in 25 mM TrisHCl pH 7.5 and 1 mM DTT at room temperature. The pooled and concentrated peak fractions, as determined by Bradford assay and SDSPAGE, were then purified on a Sephadex75 gel filtration column (Pharmacia) in 25 mM TrisHCl pH 7.5, 10 mM NaCl and 1 mM DTT. The peak fractions were concentrated and stored at -80°C in aliquots. Careful purification of the heterodimer yielded about 15 mg of clean heterodimer from 2 liters of media.
Refolding of the p50/p65 heterodimer
Truncated recombinant murine p50 RHRL (residues 39364) and p65 RHRs (residues 19291) 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 in 20 mM NaCl, 10 mM MES pH 6.0, 10 mM ßME, 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 20500 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 50400 mM NaCl in 10 mM MES pH 6.0 and 5 mM ßME 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 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 20500 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 excess of p65 and a final total protein concentration of 154666 µg/ml in 7 M urea, 0.5 M NaCl, 1.0 mM EDTA, 0.2 mM PMSF, 10 mM ßME and 25 mM TrisHCl, 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 TrisHCl pH 7.5 and 20 mM NaCl for a total of four dialysis events.
The lysate was then loaded onto a cation exchange SP-Sepharose fast flow column and the protein purified with a linear salt gradient at 20300 mM NaCl in 25 mM TrisHCl, pH 7.5. Pooled peak fractions were concentrated, aliquoted and stored at -80°C. The refolding procedure yielded 15 mg of clean heterodimer from approximately 50 mg of each protein initially used.
DNA purification
Oligonucleotides were synthesized via phosphoramidite synthesis on a Milligen Cyclone Plus DNA synthesizer. After deblocking, the oligonucleotides were purified over a Q Sepharose column on a FPLC system. Peak fractions of the desired product were pooled and immediately buffered at 50 mM MES, pH 6.0, desalted and concentrated with a 1 ml Q Sepharose column. The oligonucleotides were then desalted and concentrated in Centriprep-3 concentrators (Amicon) to approximately 2 mM in a final buffer of 10 mM TrisHCl, pH 7.5. Equimolar amounts of both strands were mixed and annealed. The final double-stranded oligonucleotide was mixed in 10% molar excess of the p50/p65 heterodimer.
Two 11 base pair double-stranded target DNAs containing the B site from the immunoglobulin kappa light chain gene were constructed each with a 5' overhang of a T on the top strand and an A on the bottom strand. 5'-GGGGACTTTCC-3' (Ig
B1); 5'-GGGACTTTCCT-3' (Ig
B2) (
B sequences boldface). One 17 base pair double-stranded blunt-ended target DNA containing the urokinase plasminogen activator
B site was constructed with the sequence 5'-CTCAGGGAAAGTACAGA-3' (uPA
B,
B sequence boldface).
Crystallization and data collection
The refolded heterodimer p50 RHRL/p65 RHRs complexed to an 11-mer DNA containing the immunoglobulin B site (Ig
B1). Crystals grew after nine days at 18°C from 6 µl hanging drops containing 6 mg/ml of the complex, 50 mM sodium acetate, pH 5.5, 100 mM CaCl2, 0.125% ß-octyl-glucopyranoside, 1 mM spermine, 8% polyethylene glycol 3350 and 10 mM DTT (Figure 5a
).
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Coexpressed heterodimer p50 RHRs/p65 RHRs was crystallized complexed to a 17-mer DNA containing the urokinase plasminogen activator B site (uPA
B). Crystals were obtained from hanging drops at 18°C in 50 mM MES, pH 6.0, 200 mM CaCl2, 0.05% ß-octyl-glucopyranoside, 0.05 mM spermine, 10 mM DTT and 8% polyethylene glycol 3350 (Figure 5b
). To confirm the presence of the proteinDNA complex in the crystal, crystals of the p50 RHRs/p65 RHRs/uPA
B complex were removed from the mother drop, washed four times in 30 µl of reservoir solution and then dissolved in sample dye. The different samples were then run on a 15% SDSPAGE gel and visualized with Coomassie stain.
Stabilizing solutions consisted of the same components as the reservoir solution except with 15% PEG 3350. Cryosolvents consisted of the same components of the stabilizing solution plus 30% ethylene glycol. Before data collection, crystals were dialyzed at 18°C for 2 days in 30 µl of stabilizing solution against 5 ml of cryosolvent. The crystals were then mounted in nylon loops and flash frozen in a liquid nitrogen stream. X-ray diffraction data of the heterodimer:IgB1 crystal were collected at 105 K using a MarResearch imaging plate system using X-rays from a large Rigaku rotation anode RU operated at 50 kV and 100 mA. The unit cell dimensions of the heterodimer bound to the Ig
B1 DNA are a = b = 106.61, c = 206.56 Å,
= ß =
= 90.0°. The space group is p43212. There is one dimer in the asymmetric unit, with a solvent volume fraction of 0.59. Structure solution is as previously described (Chen et al., 1998
). The unit cell dimensions of the heterodimer bound to the Ig
B2 DNA are a = 136.9, b = 136.1, c = 88.8 Å,
=
= 90.0°, ß = 97.5°. The space group is C2. There are two dimers in the asymmetric unit, with a solvent volume fraction of 0.43. The unit cell dimensions of the heterodimer bound to the uPA
B DNA are a = 136.21, b = 176.58, c = 96.16 Å,
= ß =
= 90.0°. The space group is C222. There is one dimer in the asymmetric unit, with a solvent volume fraction of 0.58.
Electrophoretic mobility shift assay
The oligonucleotide used for the EMSA was 5'-TCTGAGGGACTTTCCTGATC-3', which contains a high affinity heterodimer target site from the IgB light chain enhancer (boldface). This oligonucleotide was annealed to its complimentary strand and radiolabeled with 32P using T4 polynucleotide kinase (New England Biolabs). The labeled DNA was then purified using with a nucleotide removal kit (Qiagen).
Binding reactions were performed using a constant DNA concentration (2 nM) in 20 µl of binding buffer [20 mM TrisHCl (pH 8.0), 50 mM NaCl, 1 mM MgCl2, 1 mM DTT, 5 µg poly(dI-dC), 1 µg bovine serum albumin, 5% glycerol (v/v)] at 20°C for 30 min. Protein concentrations for the binding reactions were as follows: p50 RHRL, 400 nM; p50 RHRL/p65 RHRL, 40 nM; p50 RHRs/p65 RHRL, 40 nM; p50 RHRL/p65 RHRs, 40 nM; p50 RHRs/p65 RHRs, 40 nM; and p65 RHRs, 1000 nM. Higher concentrations of homodimer constructs were used due to their lower affinity for the target site. The reaction mix was then loaded onto a 6% 0.25x TBE polyacrylamide gel and run for 2 h at 120 V. The gels were then dried and exposed to autoradiography film.
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Results |
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Most commercially available expression vectors are designed for the expression of one polypeptide. With the goal of purifying the p50/p65 heterodimer, we sought to modify this expression system so that the two subunits of NFB could be expressed simultaneously. The thrombin site in the pET29b expression vector (Novagen) was replaced with a second ribosomal binding (RBS) site (Figure 1
). A PCR clone of the p50 RHRL (aa 39364) was inserted after the second RBS and tested for small scale IPTG-inducible expression. A second PCR clone of the p65 RHRs (aa 19291) was then inserted after the first RBS and also tested for small-scale expression. Other constructs were produced which contained the RHR with and without the NLS, RHRL and RHRs respectively. A total of four constructs were made: p50 RHRL/p65 RHRL, p50 RHRL/p65 RHRs, p50 RHRs/p65 RHRL and p50 RHRs/p65 RHRs (Figure 2
). Since the constructs of p65 RHR used in this experiment lack the 484 amino acid transactivation domain, the resulting molecular weight is 32 kDa.
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Since the two component polypeptides of the p50/p65 heterodimer can be purified in large amounts independently, we also attempted to obtain and purify the p50/p65 heterodimer through chemical denaturation and refolding from a mixture of the two proteins. About 4550 mg of each protein were mixed and denatured with a slight excess of p65. The heterodimer was then gradually refolded by sequential dialysis. The protein mixture was separated over a gravity flow cation exchange column and then over a FPLC cation exchange column.
Activity of NFB dimers
Gel shift assays performed with different heterodimer constructs demonstrate that the proteins bind specifically to DNA (Figure 4). Different dimers were tested with an EMSA: p50 RHRL/p50 RHRL, p50 RHRL/p65 RHRL, p50 RHRL/p65 RHRs, p50 RHRs/p65 RHRL, p50 RHRs/p65 RHRs and p65 RHRL/p65 RHRL. The smearing seen in the p65 RHRL/p65 RHRL lane is due to the inherent kinetic instability of the p65 homodimer:DNA complexes. Not only do each of the DNA bound heterodimers show an appropriate size shift relative to one another but there are also no visible contaminating bands corresponding to homodimers. The band intensities also demonstrate that the presence or absence of an NLS has no effect on the DNA affinities. EMSA results are consistently similar for refolded and expressed proteins. Fluorescence polarization experiments with proteins obtained from both methods confirm that they have the same DNA binding affinity (unpublished results).
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The p50/p65 NFB heterodimer was first discovered bound to a site in the intronic enhancer of the immunoglobulin
gene of B cells: 5'-GGGACTTTCC-3'. One of the main goals in purifying NF
B was to crystallize it on this target site. Two DNAs were designed: 5'-TGGGGACTTTCC-3' (Ig
B 1) and 5'-TGGGACTTTCCT-3' (Ig
B 2) (
B sites boldface). As studies progressed, it was decided that a crystal structure of the protein bound to a non-concensus yet biologically relevant NF
B site would also be informative. Not only would this structure reveal if the protein adopted sequence dependent conformations, but by using a longer DNA it would hopefully show to what degree the DNA itself were being altered. The heterodimer was thus also crystallized on the
B site found in the human urokinase plasminogen activator enhancer. The DNA consisted of a 5'-CTCAGGGAAAGTACAGA-3' (uPA
B).
The refolded heterodimer p50 RHRL/p65 RHRs was crystallized complexed to IB1 (Figure 5a
) and I
B2. Co-expressed heterodimer p50 RHRs/p65 RHRs was crystallized complexed to uPA
B (Figure 5b
). Crystals were obtained from hanging drops and grew in a range of conditions between 78% PEG 3350, 50 mM buffer, 100200 mM CaCl2, 0.050.125% ß-octyl-glucopyranoside and 0.050.1 mM spermine and 10 mM DTT. A crystal of the heterodimeruPA
B complex was dissolved in buffer and analyzed by SDSPAGE to confirm the presence of both subunits in the crystal (Figure 5c
). The X-ray crystal structure of the heterodimerI
B1 complex was solved using a home source collected data set that extended to 2.9 Å (Figure 6a
) (Chen,F.E. et al., 1998
). Diffraction data collected of the heterodimerI
B2 complex at a synchrotron source extended to 3.0 Å (Figure 6b
). Diffraction data for the heterodimeruPA
B complex crystal were taken at a synchrotron source at 2.3 Å (Figure 6c
). The structure for this crystal complex is still being determined.
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Discussion |
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The coexpression system has also been useful in proteinprotein interaction studies with various NFB deletion mutants. In the future this system will be useful in the preparation of NF
B heterodimers containing full-length p65, including its transactivation domain. Full length recombinant p65 is currently difficult to purify as it is generally insoluble and susceptible to degradation. It would be of great value to obtain this protein in the heterodimer form since transactivation by NF
B proteins occurs most frequently in the context of heterodimers.
Small modifications to this coexpression system also have the potential to expand the number of proteins expressed together. For instance, the insertion of a third ribosomal binding site into this expression plasmid would facilitate the production of ternary complexes. One could alternatively use a second expression vector system with a different origin of replication and express quaternary complexes. Another variation would be to employ a single plasmid with different promoters driving expression.
Within the field of protein crystallography, it is often advantageous to study proteins in the context of their binding partners. It is becoming more evident that biological events are the result of the collective interactions of various proteins, such as transcription factor enhanceosomes. Many of these interprotein contacts are mediated by domains and loops that are long and flexible and thus particularly prone to degradation. Coexpression of proteins in their relevant complexes allows these regions to be compacted into their functional form and minimizes the spatial and temporal opportunity for degradation inherent in the refolding and mixing strategies commonly seen today. A plasmid with different promoters to control the expression of certain proteins would be useful in situations where it has been demonstrated that the different components have a definite order of assembly.
There are however, limitations to this system. Obviously this procedure does not allow for eukaryotic post-translational modifications. This method of purification would also not be successful for multimers in which the desired product is not strongly thermodynamically favored over the other different possible combinations. For example, it would be difficult to purify protein heterodimers with lower association constants than those of their respective homodimers. Despite these drawbacks, we feel that this mode of purification could easily be utilized for multiprotein systems that would benefit from easy recombinant expression.
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Acknowledgments |
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Notes |
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References |
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Received October 27, 1998; revised January 26, 1999; accepted January 28, 1999.