Keap1 purification
pKeap1/PET28a(+) was transformed into bacteria BL21(DE3) (EMD Chemicals, Gibbstown, NJ). Purification of Keap1 was performed based on modification of published procedures (Hong et al., 2005b). The bacteria were first grown in 10 ml of Luria-Bertani broth medium at 37°C overnight and then transferred to a 4-liter Luria-Bertani broth with absorbance at λ600 nm adjusted to between 0.2 and 0.3. Culture was continued at 37°C with vigorous shaking until optical density at 600 nm reached between 0.5 and 0.6. Isopropyl-β-d-thiogalactoside was added, and culture was continued for another 24 h with gentle agitation at 15°C. Keap1 was purified using the nickel nitrilotriacetic acid-agarose affinity chromatography (QIAGEN). Purified Keap1 was dialyzed against 25 mM Tris-HCl, pH 8.4, containing 5 mM EDTA and 5 mM β-mercaptoethanol, and was then concentrated with Centricon 30 (Millipore Corporation, Billerica, MA) and confirmed for purity by SDS-polyacrylamide gel electrophoresis and mass spectrometry.
The expression vector containing mouse Keap1-DC with a
C-terminally fused Mxe intein/chitin-binding domain was expressed
in Escherichia coli BL21 CodonPlus (DE3)-RIL (Stratagene)
cultured in Luria Broth (LB) medium at 310 K. Overexpression of
the recombinant fusion protein was induced by adding isopropyl-�-d-
thiogalactoside (IPTG) to a final concentration of 0.5 mM and by
shifting the culture temperature to 298 K. After 18 h further incu-
bation, the cells were harvested and resuspended in 20 mM Tris±HCl
pH 8.3, 1%(v/v) Triton X-100, 1 mM EDTA, 10 mg mlÀ1 DNase I,
5 mM MgCl2, 2 mM dithiothreitol (DTT) and Complete protease
inhibitor (Roche). Cells were mechanically lysed by sonication
(Branson Sonifier 450) on ice. The soluble protein fraction was then
recovered by centrifugation at 27 000g for 30 min at 277 K. The
recombinant mouse Keap1-DC fusion protein was captured by chitin-
binding beads. The protein-bound af®nity beads were washed with 40
column volumes of buffer A (20 mM Tris±HCl pH 8.3, 1 mM EDTA
and 1 mM benzamidine±HCl) containing 0.3 M NaCl and 1% Triton
X-100, followed by 40 column volumes of buffer A. On-column
protein cleavage for the retrieval of native mouse Keap1-DC was initiated by incubating the beads for 24 h with one column volume of
buffer A in the presence of 50 mM DTT and 50 mM mercaptoethane
sulfonate (Mesna) at 277 K. The recovered mouse Keap1-DC was
further puri®ed on anion-exchange (Q2, BioRad) and Superdex S75
26/60PG columns (Pharmacia). The puri®ed mouse Keap1-DC
protein has an extra methionine at the N-terminus for translation
initiation and an extra tyrosine at the C-terminus of Cys624, which
was left over from the fusion protein after cleavage. The protein
solution was exchanged into a buffer containing 20 mM Tris±HCl pH
8.3, 20 mM DTT and 10 mM benzamidine±HCl and then concen-
trated to 4 mg mlÀ1. Finally, the protein was ̄ash-frozen and stored in
aliquots at 193 K.
Detailed Information on the Kelch-Like ECH-Associated Protein 1 (Keap1) and Neh2 Proteins Cloning, Expression, and Purification Procedures. Human Keap1 cDNA from Homo sapiens skin cells was obtained from the American Type Culture Collection (ATCC). The gene is I.M.A.G.E. (Integrated Molecular Analysis of Genomes and their Expression) Consortium CloneID 3955118 (1). The NF-E2-related factor 2 (Nrf2) gene, isolated from H. sapiens lung cells, was obtained from ATCC, I.M.A.G.E. Consortium CloneID 4548874. The genes for full-length Keap1 protein and the Neh2 domain (residues 1-97) of Nrf2 protein were directionally cloned using PCR into the pET15b vector (Novagen) for expression of an N-terminal-(His)6-tagged version of the protein. The entire gene sequence in the expression vectors was verified by dideoxy sequencing. The Keap1 protein was expressed in Escherichia coli Rosetta (DE3) pLysS cells and the Neh2 protein in BL21 (DE3) cells (Novagen) by induction with isopropyl b –d-thiogalactoside.
After expression of the proteins, cells were harvested by centrifugation, raising the Keap1 culture pH to 7.8 with 1 M Tris base before harvesting. Cell pellets were frozen at -80° C.
For the Keap1 purification, the cell pellet (15 g) was resuspended in 50 ml of Buffer A (50 mM Tris, pH 8.0/500 mM NaCl/10 mM imidazole), which also contained 7.5 mM MgSO4 , 500 m g/ml lysozyme, 3 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), a Roche Complete EDTA-free protease inhibitor tablet, and a small amount of DNase. The cells were lysed by sonication on ice and centrifuged at 40,000 ´ g. The cell lysate was loaded onto a 5-ml HiTrap column (Amersham Pharmacia Biosciences) bound with Ni2+ and equilibrated with Buffer A. Buffer B for the purification consisted of 50 mM Tris (pH 8.0), 500 mM NaCl, and 1 M imidazole. After washing the column with 5% B to remove contaminants, Keap1 was eluted in a gradient of 5 to 40% Buffer B. Fraction purity was analyzed by SDS/PAGE. Pooled fractions were dialyzed against Buffer C (10 mM NaCl/20 mM Tris, pH 8.0/2 mM DTT) at 4° C under N2. Keap1 was then loaded onto a MonoQ column (Amersham Pharmacia Biosciences) equilibrated with Buffer C, washed with Buffer C, and eluted in a 0-40% gradient of Buffer D (1 M NaCl/20 mM Tris, pH 8.0/5.5 mM DTT). Pure fractions were pooled, and buffer was exchanged by multiple concentrations using a Centricon 20, 10,000 nominal molecular weight limit (NMWL) device (Amicon) and resuspensions in exchange buffer (4 mM TCEP/21 mM Tris, pH 8.0). After determining the protein concentration, an equal volume of freezing buffer [40% (vol/vol) glycerol/29 mM Tris, pH 8.0] was added, and protein aliquots were flash frozen on dry ice and stored at -80° C. During purification and during experiments, Keap1 was kept under N2 at 4° C.
For the Neh2 purification, very similar conditions were used as for the Keap1 purification, except that the pH of the buffers was 7.5 and reducing agents were omitted. The extinction coefficients for Keap1 and Neh2 at 280 nm were determined to be 71 mM-1•cm-1 and 6.55 mM-1•cm-1 using the guanidinium HCl method of Gill and von Hippel (2).
Details on Determining the Stoichiometry of Labeling of Keap1 by Sulforaphane in the Presence and Absence of Reducing Agents. The extent of Keap1 labeling by sulforaphane in reactions used for EMSA analysis was measured by a two-step ultrafiltration procedure. First, an extinction coefficient was determined for the Keap1-sulforaphane conjugate in the absence of TCEP. In the second step, this extinction coefficient was used to quantitate the extent of modification of Keap1 by sulforaphane in the presence of TCEP.
In step 1, TCEP was completely removed from Keap1 using a Centricon-20 (10,000 NMWL) ultrafiltration device, and the resulting Keap1 was quantitated and then reacted with sulforaphane to yield seven cysteine modifications on average. The reaction was then applied to Ultrafree-MC centrifugal filter devices (5,000 NMWL), and the filtrates were analyzed for sulforaphane content. Controls without Keap1 were also analyzed. Sulforaphane-conjugated Keap1 is retained in the device, and the unreacted sulforaphane passes through the filter as filtrate. The concentration of sulforaphane in the filtrates was determined using the extinction coefficient (950 M-1•cm-1) for sulforaphane at 240 nm (3). The reaction and subsequent filtration in which Keap1 was omitted was used to determine the starting amount of sulforaphane, and subtraction of the two values yielded the amount of sulforaphane consumed in the reaction with Keap1.
The extinction coefficient of the Keap1-sulforaphane conjugate was subsequently determined by monitoring the increase in optical density (OD) of the reaction at 274 nm. Sulforaphane, even at the highest concentrations used in these experiments, has a negligible OD at 274 nm. Therefore, in the absence of TCEP, the D OD at 274 nm upon sulforaphane addition and reaction with Keap1 is due entirely to the appearance of the Keap1-sulforaphane conjugates. We determined the extinction coefficient to be 7 mM-1•cm-1 for a Keap1-sulforaphane conjugate. This number is in close agreement with the value of 8 mM-1•cm-1 at 275 nm that was determined for the glutathione (GSH)-sulforaphane conjugate (4).
In step 2, the extent of the reaction of sulforaphane with Keap1 conducted in the presence of TCEP was calculated. The OD contribution at 274 nm from the TCEP-sulforaphane conjugate in the reaction was determined using ultrafiltration and analyzing the filtrate. This contribution was subtracted from the total OD at 274 nm in the reaction, along with the absorbance of the Keap1 protein, to yield the absorbance contribution at 274 nm from the Keap1-sulforaphane conjugate. The extinction coefficient of 7 mM-1•cm-1 was used to calculate the average number of residues modified by sulforaphane per Keap1 protein.
The reaction of sulforaphane exclusively with the cysteines of Keap1 and not primary amines on residues such as lysine was verified by substituting Neh2 for Keap1. No conjugates were observed upon addition of sulforaphane to Neh2 (data not shown), which has 6% lysine content, indicating that the extinction coefficient for the Keap1-sulforaphane conjugate results from only Keap1 cysteine modification.
Details on the Modification of Keap1 with Biotinylated Iodoacetamide (BIA) and 1-Cl-2,4-Dinitrobenzene (CDNB) for Isothermal Titration Calorimetry (ITC). Keap1 protein was labeled with CDNB for 2 h on ice in a reaction containing 7 m M Keap1, 770 m M CDNB, and 20 mM Tris•HCl (pH 8.0). Residual TCEP from the Keap1 storage buffer was 0.21 mM, and residual acetonitrile from the CDNB was 0.6%. In a separate experiment, Keap1 was labeled with BIA for 70 min at room temperature in a reaction containing 19 m M Keap1, 180 m M BIA, and 20 mM Tris•HCl (pH 8.0), 0.46 mM TCEP, and 0.8% DMSO. For each experiment, unlabeled Keap1 was prepared in an identical reaction that omitted the electrophile but included the vehicle. In the reactions with CDNB, buffer was exchanged immediately to 50 mM Hepes (pH 8.0), 2 mM TCEP using a Centricon Centriplus 5000 (10,000 NMWL) device from Amicon and several washes with buffer. This method resulted in significant protein loss for the CDNB-modified Keap1, such that a dialysis method was used for the subsequent labeling of Keap1 with BIA. The reactions with BIA were quenched by adding TCEP to 3 mM, then putting on ice, followed by exhaustive dialysis at 4° C under N2 into 20 mM Hepes (pH 8.0) and 2 mM TCEP. For each experiment, Neh2 protein was exchanged to the same buffer as Keap1 by using the same method. Particulates were removed from each protein by centrifugation.
The protein concentration for BIA-modified Keap1 was determined using the extinction coefficient at 280 nm determined for nonmodified Keap1 because the conjugated biotin and linker do not contribute significantly to absorbance at 280 nm. Because the aromatic ring of CDNB will contribute significantly to the absorbance at 280 nm, the concentration of the Keap1 protein modified with CDNB was determined using amino acid analysis to be 0.44 ± 0.032 mg/ml. The mass contributions from glycine, histidine, phenylalanine, and lysine were used in the determination, and the error in the concentration in mg/ml was determined from the standard deviation in the determined masses of the four residues. The concentration of CDNB-modified Keap1 in m M was determined using the molecular weight of non-modified Keap1 (the molecular weight determined by mass spectrometry for modified Keap1 produces the same result within two significant digits). The concentration of CDNB-modified Keap1 was 2.0 ± 0.15 m M, with the error propagated from the amino acid analysis error. The number of modifications of Keap1 by CDNB was determined after removal of unreacted CDNB using the extinction coefficient of GSH-conjugated CDNB at 340 nm of 9.6 mM-1•cm-1 (5). The spectra of CDNB-modified Keap1 and non-modified Keap1 showed a difference of 0.082 absorbance units at 340 nm, corresponding to 8.6 m M conjugated CDNB molecules in the sample used for ITC. The average number of residues modified by CDNB as determined by UV absorbance is then calculated to be 4.3 ± 0.3, dividing 8.6 m M by 2.0 ± 0.15 m M.
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5. Habig, W. H., Pabst, M. J. & Jakoby, W. B. (1974) J. Biol. Chem. 249, 7130-7139.