(Received for publication, January 31, 1996; and in revised form, February 20, 1996)
From the
A major obstacle to the purification of glucocorticoid receptor (GR) is the very high nonspecific surface adsorption of this protein. This phenomenon is a property of the GR itself and does not reflect overall protein concentration or buffer conditions. We have observed that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) is unique in its ability to stabilize the receptor and largely eliminate loss to nonspecific adsorption. We have coupled this observation with a two-step purification method that allows efficient purification and stabilization of transcriptionally active glucocorticoid receptor. For this procedure, the GR first undergoes a major purification by anion exchange chromatography following hormone binding and on-column receptor transformation. Second, the GR is resolved to homogeneity utilizing a hydrophobic interaction chromatography step which consists of a 2.5 M to 0 M NaCl gradient elution of contaminating proteins followed by displacement of GR by CHAPS. GR at both stages of purification was able to activate transcription from the glucocorticoid response element containing the promoter region of the long terminal repeat of the mouse mammary tumor virus. This simple and efficient methodology should be of a considerable advantage for studies of the biology of the active, full-length GR.
The glucocorticoid receptor (GR) ()is the most
studied and best understood member of the family of steroid hormone
receptors(1) . The GR and all members of this family act as
transcription factors, either inducing or inhibiting gene expression
upon hormone binding. Glucocorticoids represent an important class of
therapeutic agents(2) , and an understanding of the functioning
of their receptor has broad implications in basic biology and the
treatment of disease.
In its inactive state the GR resides in the cytoplasm in association with chaperone proteins, 2 molecules of the 90-kDa heat shock protein, 1 molecule of the 70-kDa heat shock protein, 1 molecule of the 56-kDa heat shock protein, and a 23-kDa protein(3) . After hormone binding, some, and possibly all, of the chaperone proteins are lost (a process known as GR transformation), and GR is translocated to the nucleus(4) . Once in the nuclear domain, the GR forms homodimers and binds to DNA at specific glucocorticoid response elements (GREs), leading to activation of transcription. The GR has also been shown to interact with other transcription factors, leading to modulation of its own functioning and that of the other factors(5) . In the final step of this cyclical process, the GR is exported to the cytoplasm awaiting the next wave of activation(6) .
Multiple mechanisms for transcriptional activation by the GR have been described. Characterization at the chromatin level indicates that GR functions by some mechanism to remodel the chromatin structure of genes, facilitating the loading of transcription factors required for induction of transcription(7, 8) . GR also interacts directly, or through intermediary factors, with components of the basic transcriptional machinery(9) . These mechanisms need not be mutually exclusive, although the architecture of certain gene systems suggests displacement of GR binding prior to initiation of transcription (10) .
In vitro reconstitution studies with the intact, full-length GR have been difficult as no satisfactory protocol exists for the efficient purification of intact, stable, and biologically active GR from animal tissues and cells. Numerous approaches have been employed (for an overview, see Ref.(11) ), including the use of almost all of the typical chromatographic media, as well as affinity-based applications utilizing hormone(12, 13) , DNA(14) , and antibodies(15, 16) . Complete purification procedures have been demonstrated (14) and have contributed greatly to the understanding of GR. Nonetheless, these procedures have been hampered by low yields and instability of the purified GR.
Unfortunately, cloning of the GR has not provided an easy solution to these problems. Full-length GR is not well expressed in Escherichia coli(17) . A baculovirus expression system for active GR has been described(18) , but to date only partial purification of GR has been reported utilizing this system. Yeast systems may hold promise, as expression of a GR fusion product with glutathione S-transferase in Saccharomyces cerevisiae has been carried out(19) , but purification of active GR from this system has not been reported. Unlike the full-length protein, domains have been expressed and purified. A functional DNA binding domain of the GR can be well expressed in E. coli and the protein purified(20) . The steroid binding domain has also been expressed from a recombinant system(17) . Unfortunately this fragment has a low affinity for hormone binding. A large portion of the in vitro functional studies in the literature have, accordingly, utilized partially purified GR or have conducted examinations utilizing only the DNA binding domain of the receptor. The relationship between the activities described in these studies and that of the holo-receptor remains to be seen.
We describe here a simple and efficient method for the purification and stabilization of the GR from mammalian cells. The receptor produced by this procedure retains its biological activity and is able to activate transcription from a GRE-driven promoter. This procedure should be of a distinct advantage for studies which require the entire GR in a stable and active form.
Initial
studies utilized the glucocorticoid
[H]triamcinolone acetonide as a ligand for
following glucocorticoid binding/receptor throughout the purification.
However, as problems developed with nonspecific surface interactions of
the GR, it became advantageous to utilize a covalent ligand, which
would allow direct tracking of GR. More specifically, one could equate
loss of radioactivity with loss of receptor itself rather than
dissociation and loss of labeled hormone. The procedure described
utilized the dexamethasone derivative, [
H]DexMes
to covalently label GR(24) . A typical
[
H]DexMes binding activity and A260 profile over
the elution gradient is shown in Fig. 1. Protein immunoblotting
verified that the first peak (fractions 13-18) contained GR and
that the second peak (fractions 22-26) contained HSPn-GR
(data not shown). This procedure, utilizing our preparations, resulted
in a large purification in a single step but a homogeneous preparation
of GR was not obtained ( Fig. 2and Table 1). In addition,
the receptor was not stabilized such that it could be handled without
large losses due to nonspecific interactions.
Figure 1:
Typical elution profile of anion
exchange chromatography of GR following on-column receptor
transformation. GR was eluted from a Mono Q anion exchange column by a
linear NaCl gradient from 0 to 500 mM NaCl. The calculated
gradient would begin at fraction 8 and reach 500 mM from
fraction 26 until completion of the procedure. The conductivity
resulting from this gradient is shown by the filled circles. The filled
squares represent the absorption at 280 nM over the elution
profile. The open circles show the cpm in 25 µl of each fraction
arising from [H]DexMes-GR. Preceding elution the
Mono Q column was 1) loaded with NaMoO
stabilized cell
cytosol of GR overexpressing WCL2 cells which had been incubated with
[
H]DexMes for 16 h and brought to a final NaCl
concentration of 260 mM, 2) washed with buffer containing 260
mM NaCl and 6 mM NaMoO
, and 3) washed
with buffer containing no NaCl or NaMoO
. Addition details
are given under ``Experimental
Procedures.''
Figure 2:
Silver staining and fluorography of anion
exchange purified [H]DexMes bound GR. Peak
fractions from the anion exchange chromatography step were pooled,
concentrated and resolved by SDS-PAGE electrophoresis. Following this
the resulting protein bands were visualized by silver staining (A) or fluorography (B). The migration of molecular
mass standards is indicated on the right side of the figure. GR has a
molecular mass of 94 kDa(14) .
Losses of the GR due to nonspecific interactions during this stage of the purification were largely controlled by addition of CHAPS at a final concentration of 7.5 mM to both of the running buffers (see next section). CHAPS also had the added benefit that it consistently led to increased transformation of HSPn-GR during the purification as reflected by the relative sizes of peaks 1 and 2 (data not shown). A shorter column of larger diameter and use of maximal flow rates during the elution gradient further minimized nonspecific loss of GR. An additional modification was the loading and washing of the column at a higher salt concentration (260 mM) to further eliminate contaminating proteins.
The purity of the preparations from this
step is shown in Fig. 2. In panel A silver staining of
a SDS-PAGE gel of this material indicates the presence of 10-15
major proteins; the largest having a molecular mass of 94 kDa, the
molecular mass of GR. At least four of these proteins are fragments of
the GR as indicated by fluorography of a SDS-PAGE gel of
[H]DexMes labeled material (panel B).
These fragments are the result of either proteolysis or expression of
shorter than full-length GR. If they are the result of proteolysis, it
most likely occurred prior to the chromatography as the amount of GR or
the number and amount of the proteolytic fragments did not change
following overnight incubation of concentrated anion exchange GR at
room temperature (data not shown). Proteolysis, at least by serine
proteases, during the chromatographic separation is also unlikely since
GR or its shorter fragments were unchanged by the inclusion or
exclusion of serine protease inhibitors in the running buffers as
described in the original procedure.
A number of detergents were examined for their ability to inhibit nonspecific adsorption of GR. A list of detergents examined includes Triton X-100, Tween 20, Nonidet P-40, Lubrol PX, octylglucoside, and CHAPS. Concentrations below, equal to, and above the critical miscelle concentration were examined with and without the addition of salt and/or carrier protein. Mixtures of detergents which had been described to stabilize membrane proteins (26) were tested as well. The zwitterionic derivative of deoxycholate, CHAPS, proved to be exceptional for stabilization of GR. Addition of CHAPS to a final concentration of 10 mM minimized nonspecific loss to 20% allowing handling of the partially purified GR.
The high degree of nonspecific adsorption of the GR also made concentration of fractions difficult. GR bound avidly to dialysis membranes and concentration methods which used them resulted in complete loss of receptor under numerous conditions. Ultrafiltration apparatuses (Amicon 30; Amicon, Inc.) could be used for concentration if nonspecific binding to the membranes and polystyrene structure was blocked by BSA or insulin. In addition, CHAPS had to be present at a concentration of at least 5 mM but less than 30 mM. Utilizing these conditions, recoveries exceeding 80% could be achieved. Table 1lists the ultimate yields and fold purification following the concentration for each of the purification steps.
In an effort to utilize a
component of the nonspecific binding to advantage, chromatography of
the GR utilizing hydrophobic interaction medium was examined. Use of
this medium was not without its problems, but eventually gave way to a
useful methodology. The GR bound well to hydrophobic interaction medium
(phenyl- or butyl-Sepharose) in the presence of high salt
concentrations, but was not eluted by a typical protocol of salt
elimination. Further, receptor was not eluted from either medium by
gradients of decreasing polarity such as 0 to 30% ethylene glycol or 0
to 50% isopropanol. However, CHAPS was able to displace the receptor
from these media and was chosen for this procedure. The final method
consisted of loading of GR in 2.5 M NaCl buffer, followed by a
gradient/wash to no NaCl and elution of the receptor by a 0 to 50
mM CHAPS gradient in the presence of 100 mM NaCl. A
typical chromatographic profile is shown in Fig. 3. Utilizing
this methodology, GR was purified to homogeniety as seen in the single
94-kDa band following SDS-PAGE and silver staining or fluorography of
[H]DexMes bound receptor (Fig. 4). The
typical recovery from this step following concentration was 37% giving
an overall yield of 20% (Table 1).
Figure 3:
An elution profile typical of hydrophobic
interaction chromatography of GR. GR was displaced from the column by a
linear CHAPS gradient from 0 mM to 50 mM; the
calculated CHAPS concentration at the end of the column is indicated by
the dashed line. The filled squares represent the
absorption at 280 nM over the displacement profile. The open circles show the [H]DexMes-GR
counts/min in 25 µl of each fraction. The stages of the
chromatography prior to the CHAPS displacement were: 1) addition of
NaCl (final concentration of 2.5 M) to anion exchange purified
GR, 2) loading, 3) a 2.5 M to 0 M NaCl gradient, and
4) wash with 0 mM NaCl buffer and equilibration of the column
with 100 mM NaCl. A detailed description is provided under
``Experimental Procedures.''
Figure 4:
Silver staining and fluorography of
hydrophobic interaction purified [H]DexMes-bound
GR. Peak fractions from the hydrophobic interaction chromatography step
were pooled, concentrated and resolved by SDS-PAGE electrophoresis. The
resulting gels underwent silver staining (A) or fluorography (B) for visualization of the protein bands. The position of
molecular mass standards is indicated on the right side of the
figure. GR has a molecular mass of 94
kDa(14) .
Figure 5: GR at both stages of purification activated transcription from the long terminal repeat promoter of the mouse mammary tumor virus. Illustrated is the quantification of phosphorimages resulting from in vitro transcription reactions utilizing a G-free assay system. The templates utilized are shown in panel A. The GRE template consisted of a 380-base pair G-free cassette and the promoter region of the MMTV long terminal repeat (MMTV-LTR) from bases -334 to -23 relative to the transcription start site; this fragment contains four GREs. The TATA template consisted of the MMTV TATA box region, bases -41 to -23 relative to the transcription start site, linked to a G-free cassette. Panel B shows the activity of the anion exchange purified GR and panel C that of the hydrophobic interaction-purified GR. Fold induction was determined relative to the activity of the templates in the absence of GR. The values represent the average of at least two experiments.
HI-GR also activated transcription from the GRE template, although less vigorously than the anion exchange purified fractions (Fig. 5, panel B). A progressive increase in transcription was observed reaching a level of 2.3-fold induction at a 50 nM GR concentration. Non-GRE driven transcription was not induced by the addition of the homogeneous GR to a level of 50 nM. Again, no effect was observed with the buffer system used.
Preliminary experiments show that addition of proteins eliminated during the hydrophobic interaction step was able to partially restore activity lost during this stage (Fig. 6). A combination of the HI-GR and the salt gradient fractions (SGF), following their dialysis and concentration, led to increased DNA binding to a GRE-containing template, as monitored by electrophoretic mobility shift assay analysis (pane A), and to an increase in GRE-driven transcriptional activation (panel B). The SGF alone caused no change in DNA mobility and had an inhibitory effect on transactivation of the TATA template. The results cannot be explained by nonspecific protein stabilization, as they were not duplicated by the addition of carrier proteins (data not shown). These initial findings suggest that factors important for GR DNA binding and/or transcriptional activation are lost during HI chromatography.
Figure 6: Addition of the proteins eliminated during hydrophobic interaction chromatography increases HI-GR DNA binding and transcriptional activation. Illustrated in panel A is the phosphorimage of HI-GR binding to a consensus GRE (see ``Experimental Procedures'') template as analyzed by electrophoretic mobility shift assay. Panel B shows quantification of HI-GR transcriptional activation in the presence and absence of the SGF; the templates utilized are those described in Fig. 5. The plotted activity shows the relative change in induction in the presence of SGF versus that observed in the absence of SGF (Fig. 5).
Presented here is a methodology for the rapid and efficient purification of stable and transcriptionally active GR. It is a two-step procedure utilizing a first step of on-column transformation and anion exchange chromatography followed by hydrophobic interaction chromatography with CHAPS elution. The overall recovery of this procedure is 20% of the starting GR with a yield of 5-10 µg of protein. Although the procedure was carried out utilizing tissue culture cells as the protein source, other sources in which the GR is found untransformed in association with heat shock proteins could also be used.
The major problem encountered working with the GR was the inherent instability of transformed GR following any significant purification. In this regard, possibly the most significant result described here is the development of conditions for stabilization of the GR. Judging from our experience, this instability is likely to have been a major barrier to the study of holo-GR in the past. In the absence of stabilizer, the GR exhibited very high nonspecific binding to the surfaces of any containers with which it came into contact. The seriousness of this problem can be readily assessed by the fact that losses as high as 70% can result from mere pipetting of the receptor, using a typical polypropylene pipette tip. This effect was not simply a hydrophobic interaction or the result of dilute protein solutions. Stabilization experiments (data not shown) revealed that minor stabilization could be accomplished by the addition of carrier protein, salt, or detergent, indicating the involvement of hydrophobic, as well as hydrophillic, elements of the protein itself. Ultimately, addition of the zwitterionic detergent, CHAPS, allowed inhibition of this interaction such that handling and study of the intact protein was possible.
CHAPS also proved to have other benefits in addition to its stabilizing properties. First, its inclusion in the anion exchange stage led to increased yields. The described methodology(23) , which we used for the initial step of the purification, incorporated 2% acetonitrile in the buffer systems to aid in the washing of unbound steroid from the column. We did observe some stabilization of GR by acetonitrile and we would suggest that it decreased nonspecific adsorption of hormone-bound GR. These studies also revealed that use of CHAPS, in lieu of acetonitrile, resulted in larger yields of receptor (data not shown). This increase appeared to be the result of an increase in on-column transformation of the GR, as well as a decreased loss of GR due to nonspecific adherence. Finally, CHAPS proved to be very useful in the displacement of the GR from the hydrophobic interaction column used in the final stage of the purification.
CHAPS, in the ranges necessary for GR stabilization, did not inhibit the biological activity of the receptor as reflected by activation of transcription. Transcriptional activation was observed following the inclusion of as much as 15 mM CHAPS. This is a considerable level given that the critical miscellization concentration of this detergent is approximately 1 to 3 mM at the salt concentrations used in these assays(27) . CHAPS is known as a mild nondenaturing detergent and has a planar structure containing a hydrophobic and a hydrophilic side. CHAPS miscelles are sandwich-like in nature with aggregation numbers of less than 10 members. Interestingly, this detergent has also been described to have a unique activity for increasing binding of a number of DNA-binding proteins in gel mobility retardation assays(28) . However, there is a critical range of CHAPS concentration which can be utilized in this protocol. Step elution of the GR from the hydrophobic interaction column by CHAPS concentrations greater than 50 mM led to irreversible loss of GR transactivation activity (data not shown).
Although CHAPS was able to significantly stabilize the GR-containing fractions, it was not sufficient by itself to allow the concentration of samples using ultrafiltration devices. GR in fractions from either stage of the purification was significantly lost to nonspecific adsorption in these devices. Increases in the CHAPS concentration did little to decrease this effect but blocking of the nonspecific adsorption could be achieved by treatment of the membranes and polystyrene structures of these units with BSA. Such pretreatment led to recovery of 70-90% of the GR from either stage of the purification. Nonetheless, inclusion of CHAPS remained necessary for recovery. However, inclusion of CHAPS at concentrations greater than 30 mM led to a significant decrease in recovery from the concentrators. Possibly the effect resulted from solubilization of the passivating proteins and re-exposure of sticky surfaces. Furthermore, treatment with BSA solutions above 5 mg/ml also decreased recovery; potentially, this resulted from competition for available CHAPS miscelles during the latter part of the concentration process.
A substantial difference in the transcriptional activation was observed between the anion exchange and the hydrophobic interaction-purified GR preparations. The less pure anion exchange fractions had approximately 6-fold the activity of the homogeneous hydrophobic interaction fractions. Two potential explanations exist; either the protein is being denatured during the final purification step, or a GR-associated activation factor is missing from the homogeneous preparations. Preliminary experiments suggest the involvement of an accessory factor. Addition of proteins eliminated during the final purification step both increased GR binding to DNA and stimulated transcriptional activation. In addition, single examinations with higher concentrations of GR at both stages of the purification demonstrate achievement of maximal activation of transcription over the range of GR concentrations shown here (data not shown). This suggests that both preparations exhibit their full range of activity over a similar GR concentration span and that they differ in their intrinsic activity. This result may also be explained by the presence of an accessory protein. Procedures are currently being modified to allow the production of GR at higher concentrations and isolation of proteins eliminated during the hydrophobic interaction step for a definitive answer to this question.