- Email: [email protected]
Reconstitution of functional nuclear receptor proteins using high pressure refolding
Molecular Genetics and Metabolism 85 (2005) 318–322 www.elsevier.com/locate/ymgme
Reconstitution of functional nuclear receptor proteins using high pressure refolding Brigitte E. Schoner a, Kelli S. Bramlett a,b, Haihong Guo a, Thomas P. Burris a,b,¤ a
b
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA Received 4 March 2005; received in revised form 27 April 2005; accepted 29 April 2005 Available online 8 June 2005
Abstract Members of the nuclear receptor superfamily are ligand dependent transcription factors and many of the receptors are diYcult to produce in their functional form. Here, we describe a method for obtaining functional nuclear receptor ligand binding domain proteins from bacterial expressed inclusion bodies by high hydrostatic pressure induced refolding. High pressure refolding successfully reconstituted activity from several insoluble nuclear receptor proteins and represents a valuable tool for both functional and structural investigation of proteins or fragments thereof that might otherwise remain insoluble. 2005 Elsevier Inc. All rights reserved. Keywords: Steroid receptor; Estrogen receptor; FXR; LRH-1; Orphan receptor; Nuclear receptor; Bile acid; Protein expression
Introduction Nuclear receptors (NRs) are transcription factors that function as receptors for steroids, thyroid hormones, as well as variety of lipids. Members of this superfamily of receptors display a conserved domain structure with two highly conserved regions. The DNA binding domain is the most conserved and contains two zinc binding modules. The ligand binding domain (LBD), localized in the carboxy-terminal portion of the receptor, is the second most conserved region and is suYcient for ligand recognition and ligand-dependent transcriptional activation. Crystal structures of many NR LBDs have been characterized illustrating a conserved three-dimensional structure composed of a threelayered -helical “sandwich” [1]. Ligand recognition occurs within the interior of the receptor polypeptide to a hydrophobic binding site. Agonist binding induces a conformational change within the LBD facilitating relo*
Corresponding author. Fax: +1 317 276 1414. E-mail address: [email protected] (T.P. Burris).
1096-7192/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.04.012
cation of helix 12 of the domain allowing for formation of a functional coactivator protein recognition surface. Coactivator protein recognition and recruitment by the agonist-bound NR is essential for activation of transcription [2]. The pockets within the LBD that bind to nuclear receptor ligands, as well as the binding sites on the surface of the LBDs for coactivator LXXLL motifs tend to have signiWcant hydrophobic character. As a result, these receptors, in the absence of ligands and coactivators, tend to have regions that are prone to aggregation during puriWcation. These solubility problems can be particularly apparent when expressing NHR LBDs in bacterial systems and has necessitated the use of insect or mammalian systems in many cases. However, these systems require signiWcant resources for typically low protein yield. In contrast, bacterial expression systems utilizing multi-copy plasmids and strong, inducible promoters are most useful for obtaining high yields of recombinant proteins. However, such proteins tend to be present in inclusion bodies and are not in their native, functionally active conformation. Biologically active proteins have been
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
recovered from these inclusion bodies by solubilization in chaotropic buVer systems, followed by refolding under dilute protein concentration. Various methods for refolding have been described in the literature, but none have overcome the problems of poor recovery and the need for strong denaturants [3,4]. It has been recognized that high hydrostatic pressures can reverse protein aggregation and promote the acquisition of native structures from insoluble aggregates, even in the absence of denaturants [5,6]. Thus, conditions known to dissociate protein aggregates should be suitable for dissociating proteins from inclusion bodies. This was Wrst demonstrated by St. John et al. [5] who reported the recovery of active bacterial -lactamase from inclusion bodies. In this report, we investigated the use of high-pressure to convert the nuclear receptor for bile acids [farnesoid X receptor; FXR (NR1H4)] from an insoluble form, to a soluble, biologically active form. Furthermore, we show that the procedure successfully reconstituted the activity of two additional NRs [estrogen receptor , ER (NR 3A2) and liver receptor homolog 1, LRH1 (NR5A2)], suggesting the technique may be widely applicable to NRs and possibly other insoluble proteins.
Materials and methods High pressure reactor The components for the high pressure reactor were purchased from the High Pressure Equipment Company, Erie, Pa., and consist of a pump (PS-40), a high pressure cell (R4-6-40), and a high pressure gauge (6P650). Plasmid construction The cDNAs for FXR, LRH-1, and ER were obtained by RT-PCR ampliWcation from human liver or testis total RNA. A DNA fragment encoding the ligand binding domain of FXR (aa 242–472) was generated by PCR and was subcloned into a pET-based plasmid (Novagen, Madison, WI) that provided an N-terminal (His) 6 tag. The resulting plasmid was transformed into Escherichia coli BL21(DE3), the transformants were plated on tryptone/yeast extract (TY) agar plates containing 100 g/ml ampicillin and incubated overnight at 37 °C. LRH-1 (aa 224–495) and ER (aa 203–452 (F domain truncated)) were subcloned and expressed in a manner similar to FXR. Culture conditions Overnight cultures grown at 37 °C in TY broth (supplemented with 100 g/ml ampicillin) were diluted 1:50
319
into fresh broth and growth was continued at 37 °C until an OD600 of 0.8 was reached. IPTG (Invitrogen Life Technologies, Carlsbad, CA) was added to a Wnal concentration of 0.5 mM and growth was continued for an additional 3–5 h at which time the cells were harvested by centrifugation, and the cell pellets were stored at ¡20 °C. Isolation of inclusion bodies Inclusion bodies were isolated by diVerential centrifugation as described by Schoner et al. [7]. The Wnal pellet was resuspended in the assay buVer at concentrations indicated in the text and packaged into plastic bags and heat-sealed. Protein analysis Samples (1 ml) were removed from the E. coli culture, centrifuged, and the cell pellet was resuspended in 100 l of 2£ Tris–glycine SDS sample buVer (Invitrogen Life Technologies, Carlsbad, CA) to which urea was added to a Wnal concentration of 7 M. Lysates were mixed with an equal volume of 2£ Tris–glycine SDS/ urea sample buVer. Aliquots were heated to 95 °C for 5 min and loaded onto a 4–20% Tris–glycine polyacrylamide gradient gel (Invitrogen Life Technologies, Carlsbad, CA) and run according to the manufacturer’s instructions. The gels were stained with GelCode blue stain reagent (Pierce, Rockford, IL). The molecular weight markers were the SeeBlue Plus Prestained standards from Invitrogen Life Technologies, Carlsbad, CA. No indication of re-aggregation of any of the proteins were noted after high-pressure refolding. Proteins were stable at room temperature for at least 24 h and retained full activity after 1 year at ¡80 °C. Gel Wltration Gel Wltration was carried out with a Superdex 200 10/300 GL column (Amersham). One hundred microliter samples (5 mg/ml) were loaded in buVer containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 2.5 nM EDTA, 1 mM DTT, and 10% glycerol. The Xow rate was 0.5 ml/min. The size standards were purchased from Bio-Rad. Coactivator interaction assay The nuclear receptor coactivator interaction assay was performed as previously described for the liver X receptor [8]. For constitutively active LRH-1, the amount of His-LRH-1 LBD protein was held constant at 20 nM while the concentration of GST-coactivator protein was varied.
320
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
Results and discussion Fig. 1A shows the reactor that we designed to generate pressures up to 3 kbar. This reactor can pressurize multiple samples at a time and hold volumes of up to 1.2 L. To demonstrate the utility of this reactor, we prepared inclusion bodies of N-terminal His-tagged FXR LBD produced in E. coli as described in Materials and methods. For the high-pressure refolding, the puriWed inclusion bodies were packaged into a sealed plastic bag. After
placing the bags inside the oil-containing pressure cell, the samples were slowly pressurized to 2.5 kbar, and kept at that pressure for 16 h at room temperature. Depressurization was at a rate of 200 bars/15 min. Soluble protein present in the supernatant (following centrifugation) was analyzed for protein concentration and size. As shown in Fig. 1B, the FXR LBD is produced at very high levels following IPTG induction (lane 3) and is found exclusively in the insoluble fraction (lane 7). About 40–50% of the protein present in the inclusion bodies was recovered
Fig. 1. Refolding FXR LBD using a high-pressure generator. (A) Diagram (left panel) and photo (right panel) of the high-pressure generator. (B) Analysis of FXR LBD protein expression and puriWcation by SDS–PAGE analysis using a 4–20% gradient gel stained with GelCode (Pierce). Left panel: expression of FXR LBD in E. coli before (lane 2) and after (lane 3) induction with IPTG and the lysed cells (lane 5) were centrifuged to separate the soluble (lane 6) from the insoluble (lane 7) fractions. Markers in kDa are in lanes 1 and 4. Right panel: FXR LBD after high pressure refolding. Soluble fractions from the three concentrations: 1 mg/ml (lane 2); 5 mg/ml (lane 3), and 10 mg/ml (lane 4). The corresponding insoluble fractions are in lanes 6–8. (C) Gel Wltration of the refolded FXR LBD. The »30 kDa FXR LBD elutes as a single peak between the 17 kDa and the 44 kDa size standards.
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
in the soluble fraction following the high-pressure treatment, independent of protein concentration (lanes 2–4; right panel). The purity of the FXR LBD was greater than 95%, and it existed primarily as a monomer (Fig. 1C). Total recovery of soluble protein was 4 mg/100 ml culture. Although soluble FXR LBD has been previously obtained from E. coli by optimizing expression conditions [9], the high-pressure refolding procedure required no optimization of expression conditions and 95% purity was obtained directly from the refolding procedure without need for further puriWcation. Activity of the FXR LBD was assessed using an ampliWed luminescent proximity homogenous assay previously described [8]. This assay monitors the agonistinduced conformational change in the LBDs and depends on protein/protein interactions between the NHR LBDs and coactivators. GST-fusion proteins of the NR interacting domains from three members of the SRC-1 family of coactivators (SRC-1, SRC-2, and SRC3) facilitate monitoring of the functional state of the NR LBD within the assay [8]. As shown in Fig. 2A, soluble FXR LBD displayed the ability to ligand-dependently recruit coactivator proteins illustrating its functional reconstitution from the inclusion bodies. NRs often display preference for particular coactivator proteins [10] and this was indeed observed for FXR, with SRC-2 being recruited most eYcaciously followed by SRC-1. To determine if the high-pressure refolding method could be applied to other NR LBDs, we examined the ER LBD. Previously, we were able to obtain soluble
321
ER LBD from E. coli only through site-directed mutagenesis of the hydrophobic surface amino-acid residues. We revisited expression of the wild-type ER LBD using an identical expression system and puriWcation procedure as that used for FXR described above. Like the FXR LBD, the ER LBD was found exclusively in the insoluble fraction as a »29 kDa protein (data not shown). Following high pressure refolding, 200 g protein/ 100 ml culture was recovered in the soluble fraction and tested for activity in the biochemical coactivator recruitment assay. As shown in Fig. 2B, ER displayed functional activity demonstrating ligand-dependent recruitment of all three coactivator proteins thus conWrming successful reconstitution of functional activity. The third NR LBD examined was that of LRH-1, an orphan member of the nuclear receptor superfamily that plays a role in metabolic regulation [11]. Approximately 50% of the 48 members of the human nuclear receptor superfamily do not yet have identiWed ligands and represent a signiWcant opportunity in terms of characterizing novel physiology and pharmacology. Many of these, in contrast to steroid receptors, display signiWcant constitutive transactivation activity. Thus, we would expect these receptors to also display constitutive coactivator binding activity in vitro if successful in obtaining functional protein. Using the identical expression system to that utilized for the FXR and ER LBDs, we found that the LRH-1 LBD (»34 kDa) was also insoluble (data not shown). Soluble protein (1 mg/100 ml culture) was obtained was using the high pressure refolding procedure described for
Fig. 2. Functional activity of NR LBDs refolded using high-pressure. (A) Dose-dependent recruitment of coactivators by FXR LBD using the FXR ligand GW4064. (B) Estradiol induced recruitment of coactivators to ER. (C) LRH-1 LBD displays ligand-independent recruitment of coactivators.
322
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
the FXR and ER LBDs. LRH-1 is a constitutively active orphan NR [11], thus we expected a proWle of coactivator recruitment independent of any requirement for ligand. Recently, certain phospholipids and/or phosphatidylinositols were identiWed as putative LRH-1 ligands [12,13]; however, the ligand appears to copurify with the receptor and is diYcult to displace giving the appearance of constitutive activity, which is consistent with the constitutive coactivator binding activity that we observe. To accommodate the expected constitutive coactivator binding activity, the amount of LRH-1 LBD protein was held constant while the concentration of GST-coactivator was varied to visualize the interaction between the active LRH-1 LBD protein and the coactivators. The coactivator protein titration shown in Fig. 2C clearly indicates that the LRH-1 LBD is functionally active, constitutively binding all three coactivators independent of addition of any ligands. Thus, the high-pressure refolding method was able to reconstitute the activity of insoluble NHR LBDs into their physiologically relevant form irrespective of whether they are ligand-dependent or ligand-independent regulators of transcription. In conclusion, we have demonstrated that high pressure refolding successfully reconstitutes functional activity of three nuclear hormone receptors. The LBDs for these receptors were produced in E. coli where they formed insoluble inclusion bodies. Recovery and reconstitution of functional activity was achieved by a 2-day procedure, providing high yields and excellent purity with no requirement for column or aYnity chromatography. Furthermore, this procedure works well under varying protein concentrations and does not require the
use of denaturants. This novel method for protein refolding we used successfully to reconstitute activity of several NRs may be well suited for recovering activity of other recombinant proteins trapped in inclusion bodies.
References [1] D. Moras, H. Gronemeyer, Curr. Opin. Cell Biol. 10 (1998) 384– 391. [2] R.S. Savkur, K.S. Bramlett, D. Clawson, T.P. Burris, Vitam. Horm. 68 (2004) 145–183. [3] L.Z. Mi, F. Rastinejad, Methods Mol. Biol. 176 (2001) 81–90. [4] E.D. Clark, Curr. Opin. Biotechnol. 12 (2001) 202–207. [5] R.J. St. John, J.F. Carpenter, T.W. Randolph, Proc. Natl. Acad. Sci. USA 96 (1999) 13029–13033. [6] B.M. Gorovits, P.M. Horowitz, Biochemistry 37 (1998) 6132–6135. [7] R.G. Schoner, L.F. Ellis, B.E. Schoner, Bio-Technology 3 (1985) 151–154. [8] K.S. Bramlett, K.A. Houck, K.M. Borchert, M.S. Dowless, P. Kulanthaivel, Y. Zhang, T.P. Beyer, R. Schmidt, J.S. Thomas, L.F. Michael, R. Barr, C. Montrose, P.I. Eacho, G. Cao, T.P. Burris, J. Pharmacol. Exp. Ther. 307 (2003) 291–296. [9] L.Z. Mi, S. Devarakonda, J.M. Harp, C. Han, R. Pellicciari, T.M. Willson, S. Khorasanizadeh, F. Rastinejad, Mol. Cell 11 (2003) 1093–1100. [10] R.S. Savkur, T.P. Burris, J. Pept. Res. 63 (2004) 207–212. [11] E. Fayard, J. Auwerx, K. Schoonjans, Trends Cell Biol. 14 (2004) 250–260. [12] I.N. Krylova, E.P. Sablin, J. Moore, R.X. Xu, G.M. Waitt, J.A. MacKay, D. Juzumiene, J.M. Bynum, K. Madauss, V. Montana, L. Lebedeva, M. Suzawa, J.D. Williams, S.P. Williams, R.K. Guy, J.W. Thornton, R.J. Fletterick, T.M. Willson, H.A. Ingraham, Cell 120 (2005) 343–355. [13] E.A. Ortlund, Y. Lee, I.H. Solomon, J.M. Hager, R. SaW, Y. Choi, Z.Q. Guan, A. Tripathy, C.R.H. Raetz, D.P. McDonnell, D.D. Moore, M.R. Redinbo, Nat. Struct. Mol. Biol. 12 (2005) 357–363.
Reconstitution of functional nuclear receptor proteins using high pressure refolding Brigitte E. Schoner a, Kelli S. Bramlett a,b, Haihong Guo a, Thomas P. Burris a,b,¤ a
b
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA Received 4 March 2005; received in revised form 27 April 2005; accepted 29 April 2005 Available online 8 June 2005
Abstract Members of the nuclear receptor superfamily are ligand dependent transcription factors and many of the receptors are diYcult to produce in their functional form. Here, we describe a method for obtaining functional nuclear receptor ligand binding domain proteins from bacterial expressed inclusion bodies by high hydrostatic pressure induced refolding. High pressure refolding successfully reconstituted activity from several insoluble nuclear receptor proteins and represents a valuable tool for both functional and structural investigation of proteins or fragments thereof that might otherwise remain insoluble. 2005 Elsevier Inc. All rights reserved. Keywords: Steroid receptor; Estrogen receptor; FXR; LRH-1; Orphan receptor; Nuclear receptor; Bile acid; Protein expression
Introduction Nuclear receptors (NRs) are transcription factors that function as receptors for steroids, thyroid hormones, as well as variety of lipids. Members of this superfamily of receptors display a conserved domain structure with two highly conserved regions. The DNA binding domain is the most conserved and contains two zinc binding modules. The ligand binding domain (LBD), localized in the carboxy-terminal portion of the receptor, is the second most conserved region and is suYcient for ligand recognition and ligand-dependent transcriptional activation. Crystal structures of many NR LBDs have been characterized illustrating a conserved three-dimensional structure composed of a threelayered -helical “sandwich” [1]. Ligand recognition occurs within the interior of the receptor polypeptide to a hydrophobic binding site. Agonist binding induces a conformational change within the LBD facilitating relo*
Corresponding author. Fax: +1 317 276 1414. E-mail address: [email protected] (T.P. Burris).
1096-7192/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.04.012
cation of helix 12 of the domain allowing for formation of a functional coactivator protein recognition surface. Coactivator protein recognition and recruitment by the agonist-bound NR is essential for activation of transcription [2]. The pockets within the LBD that bind to nuclear receptor ligands, as well as the binding sites on the surface of the LBDs for coactivator LXXLL motifs tend to have signiWcant hydrophobic character. As a result, these receptors, in the absence of ligands and coactivators, tend to have regions that are prone to aggregation during puriWcation. These solubility problems can be particularly apparent when expressing NHR LBDs in bacterial systems and has necessitated the use of insect or mammalian systems in many cases. However, these systems require signiWcant resources for typically low protein yield. In contrast, bacterial expression systems utilizing multi-copy plasmids and strong, inducible promoters are most useful for obtaining high yields of recombinant proteins. However, such proteins tend to be present in inclusion bodies and are not in their native, functionally active conformation. Biologically active proteins have been
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
recovered from these inclusion bodies by solubilization in chaotropic buVer systems, followed by refolding under dilute protein concentration. Various methods for refolding have been described in the literature, but none have overcome the problems of poor recovery and the need for strong denaturants [3,4]. It has been recognized that high hydrostatic pressures can reverse protein aggregation and promote the acquisition of native structures from insoluble aggregates, even in the absence of denaturants [5,6]. Thus, conditions known to dissociate protein aggregates should be suitable for dissociating proteins from inclusion bodies. This was Wrst demonstrated by St. John et al. [5] who reported the recovery of active bacterial -lactamase from inclusion bodies. In this report, we investigated the use of high-pressure to convert the nuclear receptor for bile acids [farnesoid X receptor; FXR (NR1H4)] from an insoluble form, to a soluble, biologically active form. Furthermore, we show that the procedure successfully reconstituted the activity of two additional NRs [estrogen receptor , ER (NR 3A2) and liver receptor homolog 1, LRH1 (NR5A2)], suggesting the technique may be widely applicable to NRs and possibly other insoluble proteins.
Materials and methods High pressure reactor The components for the high pressure reactor were purchased from the High Pressure Equipment Company, Erie, Pa., and consist of a pump (PS-40), a high pressure cell (R4-6-40), and a high pressure gauge (6P650). Plasmid construction The cDNAs for FXR, LRH-1, and ER were obtained by RT-PCR ampliWcation from human liver or testis total RNA. A DNA fragment encoding the ligand binding domain of FXR (aa 242–472) was generated by PCR and was subcloned into a pET-based plasmid (Novagen, Madison, WI) that provided an N-terminal (His) 6 tag. The resulting plasmid was transformed into Escherichia coli BL21(DE3), the transformants were plated on tryptone/yeast extract (TY) agar plates containing 100 g/ml ampicillin and incubated overnight at 37 °C. LRH-1 (aa 224–495) and ER (aa 203–452 (F domain truncated)) were subcloned and expressed in a manner similar to FXR. Culture conditions Overnight cultures grown at 37 °C in TY broth (supplemented with 100 g/ml ampicillin) were diluted 1:50
319
into fresh broth and growth was continued at 37 °C until an OD600 of 0.8 was reached. IPTG (Invitrogen Life Technologies, Carlsbad, CA) was added to a Wnal concentration of 0.5 mM and growth was continued for an additional 3–5 h at which time the cells were harvested by centrifugation, and the cell pellets were stored at ¡20 °C. Isolation of inclusion bodies Inclusion bodies were isolated by diVerential centrifugation as described by Schoner et al. [7]. The Wnal pellet was resuspended in the assay buVer at concentrations indicated in the text and packaged into plastic bags and heat-sealed. Protein analysis Samples (1 ml) were removed from the E. coli culture, centrifuged, and the cell pellet was resuspended in 100 l of 2£ Tris–glycine SDS sample buVer (Invitrogen Life Technologies, Carlsbad, CA) to which urea was added to a Wnal concentration of 7 M. Lysates were mixed with an equal volume of 2£ Tris–glycine SDS/ urea sample buVer. Aliquots were heated to 95 °C for 5 min and loaded onto a 4–20% Tris–glycine polyacrylamide gradient gel (Invitrogen Life Technologies, Carlsbad, CA) and run according to the manufacturer’s instructions. The gels were stained with GelCode blue stain reagent (Pierce, Rockford, IL). The molecular weight markers were the SeeBlue Plus Prestained standards from Invitrogen Life Technologies, Carlsbad, CA. No indication of re-aggregation of any of the proteins were noted after high-pressure refolding. Proteins were stable at room temperature for at least 24 h and retained full activity after 1 year at ¡80 °C. Gel Wltration Gel Wltration was carried out with a Superdex 200 10/300 GL column (Amersham). One hundred microliter samples (5 mg/ml) were loaded in buVer containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 2.5 nM EDTA, 1 mM DTT, and 10% glycerol. The Xow rate was 0.5 ml/min. The size standards were purchased from Bio-Rad. Coactivator interaction assay The nuclear receptor coactivator interaction assay was performed as previously described for the liver X receptor [8]. For constitutively active LRH-1, the amount of His-LRH-1 LBD protein was held constant at 20 nM while the concentration of GST-coactivator protein was varied.
320
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
Results and discussion Fig. 1A shows the reactor that we designed to generate pressures up to 3 kbar. This reactor can pressurize multiple samples at a time and hold volumes of up to 1.2 L. To demonstrate the utility of this reactor, we prepared inclusion bodies of N-terminal His-tagged FXR LBD produced in E. coli as described in Materials and methods. For the high-pressure refolding, the puriWed inclusion bodies were packaged into a sealed plastic bag. After
placing the bags inside the oil-containing pressure cell, the samples were slowly pressurized to 2.5 kbar, and kept at that pressure for 16 h at room temperature. Depressurization was at a rate of 200 bars/15 min. Soluble protein present in the supernatant (following centrifugation) was analyzed for protein concentration and size. As shown in Fig. 1B, the FXR LBD is produced at very high levels following IPTG induction (lane 3) and is found exclusively in the insoluble fraction (lane 7). About 40–50% of the protein present in the inclusion bodies was recovered
Fig. 1. Refolding FXR LBD using a high-pressure generator. (A) Diagram (left panel) and photo (right panel) of the high-pressure generator. (B) Analysis of FXR LBD protein expression and puriWcation by SDS–PAGE analysis using a 4–20% gradient gel stained with GelCode (Pierce). Left panel: expression of FXR LBD in E. coli before (lane 2) and after (lane 3) induction with IPTG and the lysed cells (lane 5) were centrifuged to separate the soluble (lane 6) from the insoluble (lane 7) fractions. Markers in kDa are in lanes 1 and 4. Right panel: FXR LBD after high pressure refolding. Soluble fractions from the three concentrations: 1 mg/ml (lane 2); 5 mg/ml (lane 3), and 10 mg/ml (lane 4). The corresponding insoluble fractions are in lanes 6–8. (C) Gel Wltration of the refolded FXR LBD. The »30 kDa FXR LBD elutes as a single peak between the 17 kDa and the 44 kDa size standards.
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
in the soluble fraction following the high-pressure treatment, independent of protein concentration (lanes 2–4; right panel). The purity of the FXR LBD was greater than 95%, and it existed primarily as a monomer (Fig. 1C). Total recovery of soluble protein was 4 mg/100 ml culture. Although soluble FXR LBD has been previously obtained from E. coli by optimizing expression conditions [9], the high-pressure refolding procedure required no optimization of expression conditions and 95% purity was obtained directly from the refolding procedure without need for further puriWcation. Activity of the FXR LBD was assessed using an ampliWed luminescent proximity homogenous assay previously described [8]. This assay monitors the agonistinduced conformational change in the LBDs and depends on protein/protein interactions between the NHR LBDs and coactivators. GST-fusion proteins of the NR interacting domains from three members of the SRC-1 family of coactivators (SRC-1, SRC-2, and SRC3) facilitate monitoring of the functional state of the NR LBD within the assay [8]. As shown in Fig. 2A, soluble FXR LBD displayed the ability to ligand-dependently recruit coactivator proteins illustrating its functional reconstitution from the inclusion bodies. NRs often display preference for particular coactivator proteins [10] and this was indeed observed for FXR, with SRC-2 being recruited most eYcaciously followed by SRC-1. To determine if the high-pressure refolding method could be applied to other NR LBDs, we examined the ER LBD. Previously, we were able to obtain soluble
321
ER LBD from E. coli only through site-directed mutagenesis of the hydrophobic surface amino-acid residues. We revisited expression of the wild-type ER LBD using an identical expression system and puriWcation procedure as that used for FXR described above. Like the FXR LBD, the ER LBD was found exclusively in the insoluble fraction as a »29 kDa protein (data not shown). Following high pressure refolding, 200 g protein/ 100 ml culture was recovered in the soluble fraction and tested for activity in the biochemical coactivator recruitment assay. As shown in Fig. 2B, ER displayed functional activity demonstrating ligand-dependent recruitment of all three coactivator proteins thus conWrming successful reconstitution of functional activity. The third NR LBD examined was that of LRH-1, an orphan member of the nuclear receptor superfamily that plays a role in metabolic regulation [11]. Approximately 50% of the 48 members of the human nuclear receptor superfamily do not yet have identiWed ligands and represent a signiWcant opportunity in terms of characterizing novel physiology and pharmacology. Many of these, in contrast to steroid receptors, display signiWcant constitutive transactivation activity. Thus, we would expect these receptors to also display constitutive coactivator binding activity in vitro if successful in obtaining functional protein. Using the identical expression system to that utilized for the FXR and ER LBDs, we found that the LRH-1 LBD (»34 kDa) was also insoluble (data not shown). Soluble protein (1 mg/100 ml culture) was obtained was using the high pressure refolding procedure described for
Fig. 2. Functional activity of NR LBDs refolded using high-pressure. (A) Dose-dependent recruitment of coactivators by FXR LBD using the FXR ligand GW4064. (B) Estradiol induced recruitment of coactivators to ER. (C) LRH-1 LBD displays ligand-independent recruitment of coactivators.
322
B.E. Schoner et al. / Molecular Genetics and Metabolism 85 (2005) 318–322
the FXR and ER LBDs. LRH-1 is a constitutively active orphan NR [11], thus we expected a proWle of coactivator recruitment independent of any requirement for ligand. Recently, certain phospholipids and/or phosphatidylinositols were identiWed as putative LRH-1 ligands [12,13]; however, the ligand appears to copurify with the receptor and is diYcult to displace giving the appearance of constitutive activity, which is consistent with the constitutive coactivator binding activity that we observe. To accommodate the expected constitutive coactivator binding activity, the amount of LRH-1 LBD protein was held constant while the concentration of GST-coactivator was varied to visualize the interaction between the active LRH-1 LBD protein and the coactivators. The coactivator protein titration shown in Fig. 2C clearly indicates that the LRH-1 LBD is functionally active, constitutively binding all three coactivators independent of addition of any ligands. Thus, the high-pressure refolding method was able to reconstitute the activity of insoluble NHR LBDs into their physiologically relevant form irrespective of whether they are ligand-dependent or ligand-independent regulators of transcription. In conclusion, we have demonstrated that high pressure refolding successfully reconstitutes functional activity of three nuclear hormone receptors. The LBDs for these receptors were produced in E. coli where they formed insoluble inclusion bodies. Recovery and reconstitution of functional activity was achieved by a 2-day procedure, providing high yields and excellent purity with no requirement for column or aYnity chromatography. Furthermore, this procedure works well under varying protein concentrations and does not require the
use of denaturants. This novel method for protein refolding we used successfully to reconstitute activity of several NRs may be well suited for recovering activity of other recombinant proteins trapped in inclusion bodies.
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