- Email: [email protected]
Selective ligand purification using high-performance affinity beads
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 338 (2005) 245–252 www.elsevier.com/locate/yabio
Selective ligand puriWcation using high-performance aYnity beads Yoshihiro Ohtsua,g, Reiko Ohbaa, Yoshimasa Imamurab,g, Motoo Kobayashig, Hidetaka Hatorig, Tatsuya Zenkohg, Mamoru Hatakeyamac, Takashi Manabeg, Motohiro Hinog, Yuki Yamaguchid, Kohsuke Kataokae, Haruma Kawaguchic, Hajime Watanabef, Hiroshi Handaa,¤ a
Frontier Collaborative Research Center, Tokyo Institute of Technology, Yokohama 226-8503, Japan b Department of Chemical Engineering, Tokyo Institute of Technology, Tokyo 152-8552, Japan c Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan d Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8503, Japan e Laboratory of Molecular and Developmental Biology, Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma 630-0192, Japan f Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki 444-8585, Japan g Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Tsukuba 300-2635, Japan Received 11 September 2004 Available onilne 23 January 2005
Abstract Since the development of aYnity chromatography, aYnity puriWcation technology has been applied to many aspects of biological research, becoming an indispensable tool. EYcient strategies for the identiWcation of biologically active compounds based on biochemical speciWcity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that speciWcally interact with a target biomolecule using reverse aYnity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was eYciently puriWed in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse aYnity beads facilitated identiWcation of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were puriWed from a crude cell extract, respectively. These data indicate that reverse aYnity beads are powerful tools for identiWcation of both speciWc ligands and proteins that interact with receptor/ligand complexes. 2004 Elsevier Inc. All rights reserved. Keywords: Reverse aYnity beads; FK506; FK506-binding protein; Calcineurin; FKBP-rapamycin associated protein
Many proteins that play important roles in biological processes have been identiWed by aYnity chromatography, which is based on speciWc interactions between proteins or proteins and chemicals such as inhibitors and ligands. Since the development of aYnity chromatogra-
*
Corresponding author. Fax: +81 45 924 5145. E-mail address: [email protected] (H. Handa).
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.10.006
phy [1], it has been applied to many aspects of biological research. Once a compound with biological activity has been identiWed, it is possible, in principle, to determine its target protein by aYnity puriWcation. However, even when a speciWc biomolecule has been identiWed as a therapeutic target, methodologies leading to the identiWcation of chemicals that interact with the target biomolecule are somewhat limited and identiWcation of the chemicals is time consuming.
246
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
The identiWcation of chemicals that interact with a target protein is important both for basic research and for drug development. Improvement of test compounds to produce drugs with therapeutic eYcacy is essential given that these “lead compounds” themselves often present an unfavorable proWle with regard to stability, solubility, selectivity, and metabolism. Thus, it is often necessary to screen many compounds that are structurally related to the lead compound to obtain new compounds that have more eYcient therapeutic eVects with reduced toxicity and side eVects. Furthermore, many receptor-like proteins, including orphan nuclear receptors, whose ligands are yet unidentiWed, remain. In addition to the great interest in “chemical hunting,” it has recently become clear that the binding of chemicals to target molecules generally forms speciWc structures and induces the recruitment or dissociation of accessory proteins to exert biological eVects. Therefore, the identiWcation of proteins that interact with a protein having ligand-dependent speciWc structure has become an important issue in drug development and the development of direct ligand identiWcation technology would provide important new tools for research and drug discovery. We previously reported the application of high-performance aYnity latex beads, glycidylmethacrylate (GMA)1-covered GMA–styrene copolymer core beads (SG beads), to drug receptor identiWcation [2,3]. These latex beads have several advantages over conventional aYnity puriWcation supports. SpeciWcally, their lack of pores facilitates the eYcient removal of residual proteins and their extremely large surface area (1 g of beads has a combined surface area of 20 m [2]) results in a relatively high capacity. This enables the rapid and eYcient puriWcation of ligand- or drug-binding proteins. These high-performance aYnity beads have been used successfully for puriWcation of various proteins, including transcription factors [2], drug receptors [2], and cisplatin-damaged DNA binding proteins [3]. As these beads can rapidly identify target proteins, dissociation of ligands from their target protein is minimized. This minimized dissociation prompted us to develop a ligand identiWcation system based on these high-performance aYnity beads. Here we demonstrate the utility of this approach and show that “reverse aYnity puriWcation,” using proteinimmobilized latex beads, is a possible methodology for ligand identiWcation.
1 Abbreviations used: GMA, glycidylmethacrylate; DMF, N,N-dimethylformamide; NHS, N-hydroxysuccinimide; NP-40, Nonidet-P40; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; FKBP12, FKSO6-binding protein 12; FRAP, FKBP12/rapamycin-associated protein.
Methods Preparation of the reverse aYnity beads for protein immobilization For protein immobilization, we prepared succinyl SGNEGDEN beads (SGNEGDENS beads). SGNEG DEN beads [2] (5 mg) were suspended with 0.5 ml N,Ndimethylformamide (DMF). After Wve washes with DMF, the latex beads were resuspended with 1 ml 0.3 M succinic anhydride in DMF containing 10% triethylamine and mixed at room temperature for 15 h. After washing the beads with methanol, unreacted amino groups on the beads were blocked with 0.5 ml 0.3 M acetic anhydride in DMF containing 10% triethylamine at room temperature for 2 h. After washing with DMF and water, the beads were treated with 0.1 N NaOH at room temperature for 30 min, washed with water Wve times, and stored in water at 4 °C until use (SGNEGDENS beads). Protein immobilization to SGNEGDENS beads SGNEGDENS beads (5 mg) were washed with water, methanol and 1,4-dioxane. After washing with 1,4-dioxane Wve times, the beads were activated with 0.2 M Nhydroxysuccinimide (NHS) and 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride dissolved in 1,4-dioxane at room temperature for 2 h. NHS-activated SGNEGDENS beads (2 mg) were washed with distilled water, methanol, and ice-cold immobilization buVer [10 mM Hepes–NaOH, pH 7.9, 10% glycerol, 50 mM KCl, and 1 mM EDTA] three times each and coupled with His-tagged human recombinant FKBP12 in 400 l of immobilization buVer. The coupling reaction was carried out for 3 h at 4 °C, following which the beads were washed three times with 500 l immobilization buVer and incubated with 400 l 1 M ethanolamine (pH 8.0) at 4 °C for 24 h for masking. The protein-immobilized beads were stored at 4 °C until use. For the preparation of FKBP12-immobilized AYgel 10, 1 ml AYgel 10 was incubated with His-tagged human recombinant FKBP12 in immobilization buVer. The coupling reaction was performed for 3 h at 4 °C and unreacted NHS was masked with 1 M ethanolamine (pH 8.0). The quantity of immobilized FKBP12 was determined by HPLC analysis of amino acids cleaved with vapor-phase HCl hydrolysis. PuriWcation of compounds that speciWcally bind to reverse aYnity beads A mixture of chemicals was mixed with FKBP12immobilized beads (100g) and incubated in buVer E(+) [10 mM Tris–HCl, pH 7.4, 50 mM NaCl, 1 mM MgCl2,
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
247
1 mM CaCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Nonidet P-40 (NP-40), 1 mM dithiothreitol, and protease inhibitors (1 mM phenylmethylsulfonyl Xuoride, 1 M pepstatin, and 1 g/ml leupeptin)] (100 l) for 3 h at 4 °C with rotation. After incubation, the beads were washed three times with buVer E(+) and the bound compounds were eluted with methanol (100 l) and analyzed.
incubated with 200 l 10 g/ml FK506 or rapamycin solution in buVer E(+) containing 1% dimethyl sulfoxide for 3 h at 4 °C on a 5-rpm rotator. After incubation, the beads were washed with 200 l of buVer E(+) four times and then used for protein binding experiments.
Ligand quantiWcation based on reverse aYnity beads
A typical 200-l binding reaction consisted of the following: 200 l FKBP-depleted crude extract in 10 mM Tris–HCl, pH 7.4, 500 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM EDTA, 10% glycerol, 1% NP-40, protease inhibitors, and 400 g reverse aYnity beads which had reconstituted receptor/drug complexes on the surface. Reactions were incubated at 4 °C for 2 h on a 5-rpm rotator. After washing the beads twice in buVer E(+) and twice in buVer E(+) containing 1 M NaCl, bound proteins on the beads were eluted by boiling with SDS loading buVer and analyzed by SDS–PAGE followed by silver staining and immunoblotting.
FK506 in the eluate was measured by ELISA. Eluted extracts were also injected into a LC-MS system (mass, Micromass Q-Tof-2; LC, Agilent 1100 series; column, Waters XTerra MSC18, 2.5 m, 2.1 £ 20 mm). When we analyzed the broth extract of a FK506-producing strain and its eluate from FKBP12-immobilized beads on the HPLC system, binding buVer [10 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Brij-35, protease inhibitors] was used for incubation and wash. HPLC conditions have been described previously [4]. FKBP12-immobilized beads (100 g) were mixed with samples and biotinylated FK506 (1 pmol) in buVer E(+) (100 l) and incubated for 3 h at 4 °C with rotation. After incubation, the beads were washed three times with buVer E(+) and added to 100 l of streptavidin–HRP conjugate solution (1£/5000; Amersham Biosciences K.K.). After incubation for 30 min at 4 °C, the beads were washed three times with buVer E(+) and resuspended in 1 ml buVer E(+). This suspension (50 l) was mixed with 3,3⬘,5,5⬘-tetramethylbenzidine solution (50 l) for 10 min at room temperature and the reaction was stopped with 1 N HCl solution (50 l). HRP activity was measured by determining absorbance at 450 nm in a spectrophotometer. Preparation of cell extracts Nuclear extracts and cytoplasmic fractions of rat brain (cerebrum) and SH-SY5Y cells were prepared as previously described [5]. Both fractions were dialyzed against buVer E(+) excluding NP-40. Before being used for binding experiments, these cell extracts were incubated with FK506-immobilized beads [2] on a rotator at 5 rpm for 1 h at 4 °C to remove endogenous FK506-binding proteins. After repeating this step three times, unbound fractions were used as FKBP12depleted extracts. Crude and depleted extracts containing 10% glycerol were stable for several months at ¡80 °C. Reconstitution of receptor/drug complex on reverse aYnity beads To reconstitute the receptor/drug complex, FKBP12immobilized beads were washed with buVer E(+) and
Detection of complex-speciWc binding proteins
Results and discussion High recovery of ligand by reverse aYnity puriWcation We optimized the original latex beads (SGNGDEN) developed in our previous study [2] for reverse aYnity puriWcation by Wrst succinylating (SGNEGDENS beads) and then activating them with NHS. This method is ideal for protein coupling because it allows proteins to be coupled via primary amines under mild conditions in neutral aqueous solution (Fig. 1A). We selected FKBP12 as a model target protein because it has been well characterized pharmacologically [6]. When 5 nmol FKBP12 was incubated with 1 mg NHS-activated SGNEGDENS beads, 1.0–1.5 nmol FKBP12 was immobilized (Fig. 1B). FKBP12-immobilized latex beads (immobilized FKBP12 was 1.35 nmol/mg beads) were able to bind 1.32 nmol FK506/mg bead. This indicated that 98% of FKBP12 immobilized to latex beads retained binding ability for FK506. To compare our method with a conventional aYnity puriWcation method, we also coupled FKBP12 to AYgel 10. We used four doses of FKBP12 for the coupling so that diVerent amounts of FKBP12 were immobilized (Fig. 1B, A1–A4 for AYgel, B1–B4 for SGNEGDENS beads). As the quantities of immobilized FKBP12 on AYgel 10 were comparable to those on SGNEGDENS beads, we examined the eYciency of ligand binding and recovery using FKBP12-immobilized supports. When the same concentration of FK506 was incubated with either FKBP12-immobilized SGNEGDENS beads or FKBP12-immobilized AYgel 10, it was conWrmed that nearly all of the FKBP12immobilized SGNEGDENS beads maintained FK506
248
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 1. Preparation of FKBP12-immobilized reverse aYnity beads and its performance comparison with FKBP12-immobilized AYgel 10. (A) Preparation of FKBP12-immobilized reverse aYnity beads. SGNEGDEN beads [2] (5 mg) were succinated with 1 ml 0.3 M succinic anhydride in DMF containing 10% triethylamine (SGNEGDENS beads) and activated with 0.2 M N-hydroxysuccinimide (NHS) and 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride. NHS-activated SGNEGDENS beads (reverse aYnity beads: 2 mg) were then coupled with a protein (FKBP12). (B) Immobilization of FKBP12 to the reverse aYnity beads or AYgel 10. Coupling reaction was performed as described under Methods and immobilized FKBP12 was quantiWed by HCl hydrolysis. Four diVerent amounts of FKBP12 were coupled to each support (A1–A4 for AYgel 10, B1–B4 for SG beads). (C) EYcient ligand binding activity of the reverse aYnity beads. FK506 was puriWed using the beads prepared in (B). The mole ratio of extracted FK506 to immobilized FKBP12 is shown. (D) High recovery of ligand from the reverse aYnity beads. The recovery rate of extracted FK506 to input FK506 is indicated. Prepared beads were reacted with various dose of FK506 for 3 h at 4 °C and extracted with methanol. Extracted FK506 was determined by ELISA.
binding capacity (Fig. 1C) and that approximately 80– 100% of ligand was recovered from the SGNEGDENS beads, whereas only 18% was recovered from AYgel 10 (Fig. 1D). The lower percentage of recovery of FK506 from SGNEGDENS beads in a concentration of 10,000 nM is simply because that amount of FK506 exceeds the amount of immobilized FKBP12 (note that the recovery is proportional to the amount of immobilized FKBP12 in Fig. 1B). This result indicates that the SGNEGDENS beads have high performance to support biomolecules without losing their chemical binding activities. The low ligand recovery from AYgel-10immobilized FKBP12 demonstrates why conventional aYnity chromatography on agarose supports is not suitable for ligand puriWcation. In contrast, the highly eYcient removal of residual solutions from the reverse aYnity beads contributes to the relatively small loss of the bound ligand.
Development of a ligand quantiWcation system based on the reverse aYnity beads The high recovery of chemicals bound to a target molecule using reverse aYnity beads prompted us to establish a quantiWcation system based on this approach. To detect the speciWc binding of chemicals to FKBP12immobilized beads, we developed a competitive assay system. Biotinylated FK506 was synthesized (Fig. 2) and used for quantiWcation of FK506-like compounds. FKBP12-immobilized beads (1.2 nmol FKBP12 was immobilized to 1 mg beads) were incubated with various concentrations of FK506, rapamycin, or cyclosporin A in the presence of 1 pmol biotinylated FK506. After incubation, reacted beads were washed and incubated with streptavidin-conjugated horseradish peroxidase, and biotinylated FK506 bound to beads was evaluated by measuring peroxidase activity. FK506 and rapamycin
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 2. Ligand binding speciWcity of the FKBP12-immobilized beads Biotinylated FK506 (1 pmol) (A) was incubated with FKBP12-immobilized beads (0.1 mg) in the absence or presence of increasing amount of chemicals (B): FK506 (closed triangles), rapamycin (circles), and cyclosporin A (squares). SpeciWcally bound biotinylated FK506 was quantiWed using streptavidin–HRP. Open triangles indicate the control reaction using nonimmobilized beads and FK506 as a competitor.
both competed with biotinylated FK506 in a dosedependent manner, whereas cyclosporin A did not (Fig. 2). These results are consistent with the known properties of these ligands. Both FK506 and rapamycin bind strongly to FKBP12, although their resulting responses are diVerent [7], whereas cyclosporin A does not bind to FKBP12, although its biological eVects are similar to those of FK506. These results indicate that relative binding activities of chemicals to immobilized targets can be estimated using this system. High selectivity of ligand binding by reverse aYnity puriWcation To evaluate the speciWcity of reverse aYnity beads in ligand puriWcation, we tested whether the coated beads could selectively purify compounds from a mixture of chemicals. The chemical mixture was composed of 0.1 nmol each of acetoaminophen, atropine, caVeine, dexamethasone, methotrexate, nicotinic acid, phlorizin, thalidomide, tolbutamide, and FK506. These compounds were chosen on the basis of their structural and physicochemical diversity. This mixture was incubated with 0.1 mg FKBP12immobilized beads (1.1 nmol immobilized FKBP12/mg bead) and eluted with methanol, following which it was
249
analyzed by LC-MS. The level of FK506 in the eluate was 0.94 nmol/mg bead, and the recovery was almost equal to the extract from FK506 only (0.93 nmol/mg bead) (Fig. 3A). Rapamycin and FK506 were extracted speciWcally and competed with FK506 and biotinylated FK506 (Figs. 3C and D), whereas other compounds were not or were only slightly extracted (Fig. 3B). This result indicates that reverse aYnity beads are useful for the identiWcation of an active ligand from a mixture of compounds. We next examined whether FKBP12-immobilized beads could purify FK506 from a complex mixture of chemicals—cultured bacterial broth. FKBP12-immobilized beads (0.1 mg) were incubated with various concentrations of broth extract from a FK506-producing strain (Streptomyces tsukubaensis) in the presence of biotinylated FK506 (1 pmol). Bound biotinylated FK506 decreased in a dose-dependent manner, indicating that a FK506-like compound was present in the cultured broth extract (Fig. 4A). This result was conWrmed by HPLC. After incubation of 20 mg FKBP-immobilized beads with 0.4 ml broth extract, bound compounds were eluted with methanol and then analyzed by HPLC (Figs. 4B–E). Two peaks that bound to FKBP12-immobilized beads (Fig. 4D) were detected; both were competed out in the presence of biotinylated FK506 (Fig. 4E). By comparison with standard samples, these two peaks were identiWed as FK506 and C31-demethyl FK506. C31-demethyl FK506 is a FK506-related compound produced by this strain that binds to FKBP. The concentration of FK506 in the broth extract was estimated to be 75 M, which was similar to the value obtained by ELISA (82 M). Thus, these data demonstrate that the reverse aYnity bead screening system is applicable to semiquantitative extraction and that compounds that speciWcally bind to a biomolecule can be puriWed and identiWed from a complex mixture in a single step. Direct puriWcation of ligand/receptor complex binding proteins Having shown that reverse aYnity beads were capable of purifying ligands from complex mixtures, we next examined whether ligand/receptor complex binding proteins could be identiWed by this approach. As is well known, ligand/receptor complex has unique structure, which may induce the recruitment of other accessory proteins to exert biological eVects. When FK506 binds to FKBP12, the FKBP12/FK506 complex associates with calcineurin A and inhibits its phosphatase activity [8–10]. When rapamycin binds to FKBP12, the FKBP12/ rapamycin complex binds to FKBP12/rapamycin-associated protein (FRAP; also known as m-TOR and RAFT1) and inhibits the serine/threonine kinase activity of FRAP [11,12]. In both examples, a ligand-speciWc structure is essential for the binding of the ligand/recep-
250
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 3. Selective puriWcation of FKBP12 binding compounds from a crude chemical mixture using reverse aYnity beads. (A) EYcient binding of FK506 to the reverse aYnity beads. The mixture of 10 compounds containing FK506 or FK506 alone (0.1 nmol) was incubated with 0.1 mg of FKBP12-immobilized beads. The eluate was quantiWed by ELISA for FK506. All data are shown as mean § SE. (B) Selective binding of FK506 to the reverse aYnity beads. The mixture of 10 compounds containing FK506 (TIC is shown at top) was incubated with 1 mg of FKBP12-immobilized beads and the TIC of the eluate is shown at bottom. (C) ConWrmation of FK506 puriWcation by LC-MS. The same data as in (B) are shown as an ion chromatogram of FK506 in (Ca). This speciWc binding was absent in the presence of a speciWc competitor, biotinylated-FK506 (Cb). (D) Selective binding of rapamycin to the reverse aYnity beads. Selective puriWcation of rapamycin was also examined from 10 compounds containing rapamycin. The eluate was analyzed by LC-MS. Da, no competitor; Db, FK506 was used as a competitor; Dc, biotinylated-FK506 was used as a competitor.
tor complex to the binding proteins [13–17] to exert biological eVects. We next reconstituted the FKBP12/FK506 complex on beads and asked whether reverse aYnity beads can purify ligand/receptor complex binding proteins in a ligand-dependent manner. FKBP12-immobilized beads were incubated with 10 g/ml FK506 for 3 h at 4 °C. The beads were then incubated with FKBP-depleted rat brain nuclear extract and binding proteins were analyzed by SDS–PAGE. When the FKBP12/FK506 complex was used for reverse aYnity puriWcation, a complex-speciWc binding protein of molecular weight 60 kDa was puriWed (Fig. 5A, lane 5, indicated by asterisk). MS-MS analysis of trypsin-digested fragments of the binding protein conWrmed it to be calcineurin A (data not shown). This was also conWrmed by immunoblotting with anticalcineurin A (Fig. 5A, below). Immunoblot analysis suggested that approximately 20% of calcineurin A in the crude extract was puriWed from the reverse aYnity beads. In contrast, calcineurin A could not be puriWed by FKBP12-immobilized beads in the absence of FK506 (Fig. 5A, lane 4).
FKBP12-immobilized beads were incubated with rapamycin to reconstitute the FKBP12/rapamycin complex. After washing, the beads were incubated with a FKBP-depleted SH-SY5Y cytoplasmic fraction. Using this approach, a protein with a molecular weight of 270 kDa was detected as a speciWc binding protein for the reconstituted FKBP12/rapamycin complex (Fig. 5B, lane 2). MS-MS analysis of tryptic-digested fragments derived from the 270-kDa binding protein conWrmed it to be FRAP (data not shown). Immunoblot analysis also conWrmed that the 270-kDa binding protein was FRAP. Furthermore, calcineurin A was also detected in this extract using FKBP12/FK506 complex (Fig. 5B, lane 3). Taken together, the results shown above indicate that our reverse aYnity bead approach eYciently puriWes ligands and interacting proteins from complex mixtures in a single step. The relatively small amount of beads required for each experiment (»20 mg of beads/assay) suggests that it will be readily possible to adapt this method for high-throughput screening. After immobilization of the biomolecules of interest to the beads, they can be put in a multiwell plate, and fermentation broth,
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 4. One-step puriWcation of FK506-like compounds from cultured broth using reverse aYnity beads. (A) Various concentrations of broth extract from a FK506-producing strain (Streptomyces tsukubaensis) were reacted with FKBP12-immobilized beads (0.1 mg) in the presence of biotinylated FK506 (1 pmol). Bound biotinylated FK506 was detected using streptavidin–HRP. (B–E) HPLC analysis of the eluate from FKBP12-immobilized beads (20 mg) reacted with the cultured broth extract of the FK506-producing strain (0.4 ml). (B) Original broth extract; (C) eluate from control beads reacted with the broth extract; (D,E) eluate from FKBP12-immobilized beads reacted with the broth extract without D or with E biotinylated FK506.
a sponge extract, or chemical mixtures can be screened. Although there are several technologies that can monitor interaction between biomolecules and chemicals such as surface plasmon resonance and Xuorescence anisotropy, they are mainly designed only for analysis. As the beads developed in this study can recover the binding chemicals, they can be applied not only for analytical scales but also for preparative scales. In addition, reverse aYnity beads can identify ligand-dependent complex binding proteins, which is essential for drug development. This indicates that reverse aYnity beads can serve as a powerful tool for the screening of ligands, functional analysis of receptors, and signal transduction processes, all of which are important components for the development of new drugs. Based on this study, further evaluation of the beads in kinetics using other chemicals
251
Fig. 5. Direct puriWcation of ligand/receptor complex binding proteins using FKBP12/drug complex reverse aYnity beads. (A) PuriWcation of ligand-dependent binding protein using FKBP12/FK506 reverse aYnity beads. FKBP12/FK506 complex was reconstituted on FKBP12immobilized beads and incubated with rat brain nuclear extract and the eluate was analyzed by SDS–PAGE (lane 5) followed by silver staining (top) and immunoblot (bottom) analysis. Lane 1, 50 ng (for silver staining) or 20 ng (for immunoblotting) recombinant bovine calcineurin A; lane 2, control beads; lane 3, control beads preincubated with FK506; lane 4, FKBP12-immobilized beads; lane 5, FKBP12immobilized beads preincubated with FK506. An asterisk indicates the 60-kDa binding protein speciWcally bound to FKBP12/FK506 complex (Cal-A). (B) DiVerent ligands can purify distinct binding proteins using complex-reconstituted reverse aYnity beads. FKBP12/rapamycin complex and FKBP12/FK506 complex were reconstituted on FKBP12-immobilized beads and incubated with cytoplasm fraction of SH-SY5Y cells and the eluates were analyzed by SDS–PAGE. Lane 1, FKBP12-immobilized beads; lane 2, FKBP12-immobilized beads + rapamycin; lane 3, FKBP12-immobilized beads + FK506. Asterisks indicate speciWc binding proteins.
and proteins may be important to grasp the capability of the beads. IdentiWcation of ligands that act on receptors is one of the intriguing issues for drug development and in this study we showed that the “high-performance aYnity beads” can be used for ligand identiWcation and puriWcation. Since the development of aYnity puriWcation several decades ago, biochemists have puriWed only proteins because of technical limitations. Our study indicates that this technology can be reversed and shows that ligands can be puriWed by the “reverse” aYnity puriWcation.
Acknowledgments We are grateful to Fujisawa Pharmaceutical Co., Ltd. for providing FK506, rapamycin, cyclosporin A, FK506 ELISA system, and rat brain lysates and to M. Matsuura
252
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
and N. Shigematsu, Fujisawa Pharmaceutical Co., Ltd., for HPLC and LC-MS analysis. We are also grateful to Y. Tsuboi and to J. Kato. We thank Professor Peter K. Vogt, Scripps Research Institute for helpful discussions. We also thank Dr. Bruce Blumberg, Department of Developmental and Cell Biology, University of California at Irvine for a critical reading of the manuscript. This work was supported by a grant of R and D Projects in Cooperation with Academic Institutions from New Energy and Industrial Technology Development Organization (NEDO). R.O. is a NEDO Fellow.
References [1] L.S. Lerman, Antibody chromatography on an immunologically speciWc adsorbent, Proc. Natl. Acad. Sci. USA 39 (1953) 232– 236. [2] N. Shimizu, et al., High-performance aYnity beads for identifying drug receptors, Nat. Biotechnol. 18 (2000) 877–881. [3] T. Tomohiro, et al., Total analysis and puriWcation of cellular proteins binding to cisplatin-damaged DNA using submicron beads, Bioconjug. Chem. 13 (2002) 163–166. [4] T. Akashi, T. Nefuji, M. Yoshida, J. Hosoda, Quantitative determination of tautomeric FK506 by reversed-phase liquid chromatography, J. Pharm. Biomed. Anal. 14 (1996) 339–346. [5] J.D. Dignam, R.M. Lebovitz, R.G. Roeder, Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res. 11 (1983) 1475–1489.
[6] F.J. Dumont, FK506, an immunosuppressant targeting calcineurin function, Curr. Med. Chem. 7 (2000) 731–748. [7] W.H. Flanagan, B. Corthesy, R.J. Bram, G.R. Crabtree, Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A, Nature 352 (1991) 803–807. [8] S.L. Schreiber, Chemistry and biology of the immunophilins and their immunosuppressive ligands, Science 251 (1991) 283–287. [9] J. Liu, et al., Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes, Cell 66 (1991) 807–815. [10] N.A. Clipstone, G.R. Crabtree, IdentiWcation of calcineurin as a key signalling enzyme in T-lymphocyte activation, Nature 357 (1992) 695–697. [11] K.E. Hentges, et al., FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse, Proc. Natl. Acad. Sci. USA 98 (2001) 13796–13801. [12] A-C. Gingras, B. Raught, N. Sonenberg, Regulation of translation initiation by FRAP/mTOR, Genes Dev. 15 (2001) 807–826. [13] J.P. GriYth, et al., X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex, Cell 82 (1995) 507–522. [14] C.R. Kissinger, et al., Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex, Nature 378 (1995) 641–644. [15] J. Choi, J. Chen, S.L. Schreiber, J. Clardy, Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP, Science 273 (1996) 239–241. [16] J. Liang, J. Choi, J. Clardy, ReWned structure of the FKBP12rapamycin-FRB ternary complex at 2.2 Å resolution, Acta. Crystallogr. D 55 (1999) 736–744. [17] G.R. Crabtree, S.L. Schreiber, Three-part inventions: intracellular signaling and induced proximity, Trends Biochem. Sci. 21 (1996) 418–422.
Selective ligand puriWcation using high-performance aYnity beads Yoshihiro Ohtsua,g, Reiko Ohbaa, Yoshimasa Imamurab,g, Motoo Kobayashig, Hidetaka Hatorig, Tatsuya Zenkohg, Mamoru Hatakeyamac, Takashi Manabeg, Motohiro Hinog, Yuki Yamaguchid, Kohsuke Kataokae, Haruma Kawaguchic, Hajime Watanabef, Hiroshi Handaa,¤ a
Frontier Collaborative Research Center, Tokyo Institute of Technology, Yokohama 226-8503, Japan b Department of Chemical Engineering, Tokyo Institute of Technology, Tokyo 152-8552, Japan c Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan d Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8503, Japan e Laboratory of Molecular and Developmental Biology, Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma 630-0192, Japan f Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki 444-8585, Japan g Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Tsukuba 300-2635, Japan Received 11 September 2004 Available onilne 23 January 2005
Abstract Since the development of aYnity chromatography, aYnity puriWcation technology has been applied to many aspects of biological research, becoming an indispensable tool. EYcient strategies for the identiWcation of biologically active compounds based on biochemical speciWcity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that speciWcally interact with a target biomolecule using reverse aYnity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was eYciently puriWed in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse aYnity beads facilitated identiWcation of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were puriWed from a crude cell extract, respectively. These data indicate that reverse aYnity beads are powerful tools for identiWcation of both speciWc ligands and proteins that interact with receptor/ligand complexes. 2004 Elsevier Inc. All rights reserved. Keywords: Reverse aYnity beads; FK506; FK506-binding protein; Calcineurin; FKBP-rapamycin associated protein
Many proteins that play important roles in biological processes have been identiWed by aYnity chromatography, which is based on speciWc interactions between proteins or proteins and chemicals such as inhibitors and ligands. Since the development of aYnity chromatogra-
*
Corresponding author. Fax: +81 45 924 5145. E-mail address: [email protected] (H. Handa).
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.10.006
phy [1], it has been applied to many aspects of biological research. Once a compound with biological activity has been identiWed, it is possible, in principle, to determine its target protein by aYnity puriWcation. However, even when a speciWc biomolecule has been identiWed as a therapeutic target, methodologies leading to the identiWcation of chemicals that interact with the target biomolecule are somewhat limited and identiWcation of the chemicals is time consuming.
246
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
The identiWcation of chemicals that interact with a target protein is important both for basic research and for drug development. Improvement of test compounds to produce drugs with therapeutic eYcacy is essential given that these “lead compounds” themselves often present an unfavorable proWle with regard to stability, solubility, selectivity, and metabolism. Thus, it is often necessary to screen many compounds that are structurally related to the lead compound to obtain new compounds that have more eYcient therapeutic eVects with reduced toxicity and side eVects. Furthermore, many receptor-like proteins, including orphan nuclear receptors, whose ligands are yet unidentiWed, remain. In addition to the great interest in “chemical hunting,” it has recently become clear that the binding of chemicals to target molecules generally forms speciWc structures and induces the recruitment or dissociation of accessory proteins to exert biological eVects. Therefore, the identiWcation of proteins that interact with a protein having ligand-dependent speciWc structure has become an important issue in drug development and the development of direct ligand identiWcation technology would provide important new tools for research and drug discovery. We previously reported the application of high-performance aYnity latex beads, glycidylmethacrylate (GMA)1-covered GMA–styrene copolymer core beads (SG beads), to drug receptor identiWcation [2,3]. These latex beads have several advantages over conventional aYnity puriWcation supports. SpeciWcally, their lack of pores facilitates the eYcient removal of residual proteins and their extremely large surface area (1 g of beads has a combined surface area of 20 m [2]) results in a relatively high capacity. This enables the rapid and eYcient puriWcation of ligand- or drug-binding proteins. These high-performance aYnity beads have been used successfully for puriWcation of various proteins, including transcription factors [2], drug receptors [2], and cisplatin-damaged DNA binding proteins [3]. As these beads can rapidly identify target proteins, dissociation of ligands from their target protein is minimized. This minimized dissociation prompted us to develop a ligand identiWcation system based on these high-performance aYnity beads. Here we demonstrate the utility of this approach and show that “reverse aYnity puriWcation,” using proteinimmobilized latex beads, is a possible methodology for ligand identiWcation.
1 Abbreviations used: GMA, glycidylmethacrylate; DMF, N,N-dimethylformamide; NHS, N-hydroxysuccinimide; NP-40, Nonidet-P40; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; FKBP12, FKSO6-binding protein 12; FRAP, FKBP12/rapamycin-associated protein.
Methods Preparation of the reverse aYnity beads for protein immobilization For protein immobilization, we prepared succinyl SGNEGDEN beads (SGNEGDENS beads). SGNEG DEN beads [2] (5 mg) were suspended with 0.5 ml N,Ndimethylformamide (DMF). After Wve washes with DMF, the latex beads were resuspended with 1 ml 0.3 M succinic anhydride in DMF containing 10% triethylamine and mixed at room temperature for 15 h. After washing the beads with methanol, unreacted amino groups on the beads were blocked with 0.5 ml 0.3 M acetic anhydride in DMF containing 10% triethylamine at room temperature for 2 h. After washing with DMF and water, the beads were treated with 0.1 N NaOH at room temperature for 30 min, washed with water Wve times, and stored in water at 4 °C until use (SGNEGDENS beads). Protein immobilization to SGNEGDENS beads SGNEGDENS beads (5 mg) were washed with water, methanol and 1,4-dioxane. After washing with 1,4-dioxane Wve times, the beads were activated with 0.2 M Nhydroxysuccinimide (NHS) and 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride dissolved in 1,4-dioxane at room temperature for 2 h. NHS-activated SGNEGDENS beads (2 mg) were washed with distilled water, methanol, and ice-cold immobilization buVer [10 mM Hepes–NaOH, pH 7.9, 10% glycerol, 50 mM KCl, and 1 mM EDTA] three times each and coupled with His-tagged human recombinant FKBP12 in 400 l of immobilization buVer. The coupling reaction was carried out for 3 h at 4 °C, following which the beads were washed three times with 500 l immobilization buVer and incubated with 400 l 1 M ethanolamine (pH 8.0) at 4 °C for 24 h for masking. The protein-immobilized beads were stored at 4 °C until use. For the preparation of FKBP12-immobilized AYgel 10, 1 ml AYgel 10 was incubated with His-tagged human recombinant FKBP12 in immobilization buVer. The coupling reaction was performed for 3 h at 4 °C and unreacted NHS was masked with 1 M ethanolamine (pH 8.0). The quantity of immobilized FKBP12 was determined by HPLC analysis of amino acids cleaved with vapor-phase HCl hydrolysis. PuriWcation of compounds that speciWcally bind to reverse aYnity beads A mixture of chemicals was mixed with FKBP12immobilized beads (100g) and incubated in buVer E(+) [10 mM Tris–HCl, pH 7.4, 50 mM NaCl, 1 mM MgCl2,
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
247
1 mM CaCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Nonidet P-40 (NP-40), 1 mM dithiothreitol, and protease inhibitors (1 mM phenylmethylsulfonyl Xuoride, 1 M pepstatin, and 1 g/ml leupeptin)] (100 l) for 3 h at 4 °C with rotation. After incubation, the beads were washed three times with buVer E(+) and the bound compounds were eluted with methanol (100 l) and analyzed.
incubated with 200 l 10 g/ml FK506 or rapamycin solution in buVer E(+) containing 1% dimethyl sulfoxide for 3 h at 4 °C on a 5-rpm rotator. After incubation, the beads were washed with 200 l of buVer E(+) four times and then used for protein binding experiments.
Ligand quantiWcation based on reverse aYnity beads
A typical 200-l binding reaction consisted of the following: 200 l FKBP-depleted crude extract in 10 mM Tris–HCl, pH 7.4, 500 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM EDTA, 10% glycerol, 1% NP-40, protease inhibitors, and 400 g reverse aYnity beads which had reconstituted receptor/drug complexes on the surface. Reactions were incubated at 4 °C for 2 h on a 5-rpm rotator. After washing the beads twice in buVer E(+) and twice in buVer E(+) containing 1 M NaCl, bound proteins on the beads were eluted by boiling with SDS loading buVer and analyzed by SDS–PAGE followed by silver staining and immunoblotting.
FK506 in the eluate was measured by ELISA. Eluted extracts were also injected into a LC-MS system (mass, Micromass Q-Tof-2; LC, Agilent 1100 series; column, Waters XTerra MSC18, 2.5 m, 2.1 £ 20 mm). When we analyzed the broth extract of a FK506-producing strain and its eluate from FKBP12-immobilized beads on the HPLC system, binding buVer [10 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Brij-35, protease inhibitors] was used for incubation and wash. HPLC conditions have been described previously [4]. FKBP12-immobilized beads (100 g) were mixed with samples and biotinylated FK506 (1 pmol) in buVer E(+) (100 l) and incubated for 3 h at 4 °C with rotation. After incubation, the beads were washed three times with buVer E(+) and added to 100 l of streptavidin–HRP conjugate solution (1£/5000; Amersham Biosciences K.K.). After incubation for 30 min at 4 °C, the beads were washed three times with buVer E(+) and resuspended in 1 ml buVer E(+). This suspension (50 l) was mixed with 3,3⬘,5,5⬘-tetramethylbenzidine solution (50 l) for 10 min at room temperature and the reaction was stopped with 1 N HCl solution (50 l). HRP activity was measured by determining absorbance at 450 nm in a spectrophotometer. Preparation of cell extracts Nuclear extracts and cytoplasmic fractions of rat brain (cerebrum) and SH-SY5Y cells were prepared as previously described [5]. Both fractions were dialyzed against buVer E(+) excluding NP-40. Before being used for binding experiments, these cell extracts were incubated with FK506-immobilized beads [2] on a rotator at 5 rpm for 1 h at 4 °C to remove endogenous FK506-binding proteins. After repeating this step three times, unbound fractions were used as FKBP12depleted extracts. Crude and depleted extracts containing 10% glycerol were stable for several months at ¡80 °C. Reconstitution of receptor/drug complex on reverse aYnity beads To reconstitute the receptor/drug complex, FKBP12immobilized beads were washed with buVer E(+) and
Detection of complex-speciWc binding proteins
Results and discussion High recovery of ligand by reverse aYnity puriWcation We optimized the original latex beads (SGNGDEN) developed in our previous study [2] for reverse aYnity puriWcation by Wrst succinylating (SGNEGDENS beads) and then activating them with NHS. This method is ideal for protein coupling because it allows proteins to be coupled via primary amines under mild conditions in neutral aqueous solution (Fig. 1A). We selected FKBP12 as a model target protein because it has been well characterized pharmacologically [6]. When 5 nmol FKBP12 was incubated with 1 mg NHS-activated SGNEGDENS beads, 1.0–1.5 nmol FKBP12 was immobilized (Fig. 1B). FKBP12-immobilized latex beads (immobilized FKBP12 was 1.35 nmol/mg beads) were able to bind 1.32 nmol FK506/mg bead. This indicated that 98% of FKBP12 immobilized to latex beads retained binding ability for FK506. To compare our method with a conventional aYnity puriWcation method, we also coupled FKBP12 to AYgel 10. We used four doses of FKBP12 for the coupling so that diVerent amounts of FKBP12 were immobilized (Fig. 1B, A1–A4 for AYgel, B1–B4 for SGNEGDENS beads). As the quantities of immobilized FKBP12 on AYgel 10 were comparable to those on SGNEGDENS beads, we examined the eYciency of ligand binding and recovery using FKBP12-immobilized supports. When the same concentration of FK506 was incubated with either FKBP12-immobilized SGNEGDENS beads or FKBP12-immobilized AYgel 10, it was conWrmed that nearly all of the FKBP12immobilized SGNEGDENS beads maintained FK506
248
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 1. Preparation of FKBP12-immobilized reverse aYnity beads and its performance comparison with FKBP12-immobilized AYgel 10. (A) Preparation of FKBP12-immobilized reverse aYnity beads. SGNEGDEN beads [2] (5 mg) were succinated with 1 ml 0.3 M succinic anhydride in DMF containing 10% triethylamine (SGNEGDENS beads) and activated with 0.2 M N-hydroxysuccinimide (NHS) and 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride. NHS-activated SGNEGDENS beads (reverse aYnity beads: 2 mg) were then coupled with a protein (FKBP12). (B) Immobilization of FKBP12 to the reverse aYnity beads or AYgel 10. Coupling reaction was performed as described under Methods and immobilized FKBP12 was quantiWed by HCl hydrolysis. Four diVerent amounts of FKBP12 were coupled to each support (A1–A4 for AYgel 10, B1–B4 for SG beads). (C) EYcient ligand binding activity of the reverse aYnity beads. FK506 was puriWed using the beads prepared in (B). The mole ratio of extracted FK506 to immobilized FKBP12 is shown. (D) High recovery of ligand from the reverse aYnity beads. The recovery rate of extracted FK506 to input FK506 is indicated. Prepared beads were reacted with various dose of FK506 for 3 h at 4 °C and extracted with methanol. Extracted FK506 was determined by ELISA.
binding capacity (Fig. 1C) and that approximately 80– 100% of ligand was recovered from the SGNEGDENS beads, whereas only 18% was recovered from AYgel 10 (Fig. 1D). The lower percentage of recovery of FK506 from SGNEGDENS beads in a concentration of 10,000 nM is simply because that amount of FK506 exceeds the amount of immobilized FKBP12 (note that the recovery is proportional to the amount of immobilized FKBP12 in Fig. 1B). This result indicates that the SGNEGDENS beads have high performance to support biomolecules without losing their chemical binding activities. The low ligand recovery from AYgel-10immobilized FKBP12 demonstrates why conventional aYnity chromatography on agarose supports is not suitable for ligand puriWcation. In contrast, the highly eYcient removal of residual solutions from the reverse aYnity beads contributes to the relatively small loss of the bound ligand.
Development of a ligand quantiWcation system based on the reverse aYnity beads The high recovery of chemicals bound to a target molecule using reverse aYnity beads prompted us to establish a quantiWcation system based on this approach. To detect the speciWc binding of chemicals to FKBP12immobilized beads, we developed a competitive assay system. Biotinylated FK506 was synthesized (Fig. 2) and used for quantiWcation of FK506-like compounds. FKBP12-immobilized beads (1.2 nmol FKBP12 was immobilized to 1 mg beads) were incubated with various concentrations of FK506, rapamycin, or cyclosporin A in the presence of 1 pmol biotinylated FK506. After incubation, reacted beads were washed and incubated with streptavidin-conjugated horseradish peroxidase, and biotinylated FK506 bound to beads was evaluated by measuring peroxidase activity. FK506 and rapamycin
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 2. Ligand binding speciWcity of the FKBP12-immobilized beads Biotinylated FK506 (1 pmol) (A) was incubated with FKBP12-immobilized beads (0.1 mg) in the absence or presence of increasing amount of chemicals (B): FK506 (closed triangles), rapamycin (circles), and cyclosporin A (squares). SpeciWcally bound biotinylated FK506 was quantiWed using streptavidin–HRP. Open triangles indicate the control reaction using nonimmobilized beads and FK506 as a competitor.
both competed with biotinylated FK506 in a dosedependent manner, whereas cyclosporin A did not (Fig. 2). These results are consistent with the known properties of these ligands. Both FK506 and rapamycin bind strongly to FKBP12, although their resulting responses are diVerent [7], whereas cyclosporin A does not bind to FKBP12, although its biological eVects are similar to those of FK506. These results indicate that relative binding activities of chemicals to immobilized targets can be estimated using this system. High selectivity of ligand binding by reverse aYnity puriWcation To evaluate the speciWcity of reverse aYnity beads in ligand puriWcation, we tested whether the coated beads could selectively purify compounds from a mixture of chemicals. The chemical mixture was composed of 0.1 nmol each of acetoaminophen, atropine, caVeine, dexamethasone, methotrexate, nicotinic acid, phlorizin, thalidomide, tolbutamide, and FK506. These compounds were chosen on the basis of their structural and physicochemical diversity. This mixture was incubated with 0.1 mg FKBP12immobilized beads (1.1 nmol immobilized FKBP12/mg bead) and eluted with methanol, following which it was
249
analyzed by LC-MS. The level of FK506 in the eluate was 0.94 nmol/mg bead, and the recovery was almost equal to the extract from FK506 only (0.93 nmol/mg bead) (Fig. 3A). Rapamycin and FK506 were extracted speciWcally and competed with FK506 and biotinylated FK506 (Figs. 3C and D), whereas other compounds were not or were only slightly extracted (Fig. 3B). This result indicates that reverse aYnity beads are useful for the identiWcation of an active ligand from a mixture of compounds. We next examined whether FKBP12-immobilized beads could purify FK506 from a complex mixture of chemicals—cultured bacterial broth. FKBP12-immobilized beads (0.1 mg) were incubated with various concentrations of broth extract from a FK506-producing strain (Streptomyces tsukubaensis) in the presence of biotinylated FK506 (1 pmol). Bound biotinylated FK506 decreased in a dose-dependent manner, indicating that a FK506-like compound was present in the cultured broth extract (Fig. 4A). This result was conWrmed by HPLC. After incubation of 20 mg FKBP-immobilized beads with 0.4 ml broth extract, bound compounds were eluted with methanol and then analyzed by HPLC (Figs. 4B–E). Two peaks that bound to FKBP12-immobilized beads (Fig. 4D) were detected; both were competed out in the presence of biotinylated FK506 (Fig. 4E). By comparison with standard samples, these two peaks were identiWed as FK506 and C31-demethyl FK506. C31-demethyl FK506 is a FK506-related compound produced by this strain that binds to FKBP. The concentration of FK506 in the broth extract was estimated to be 75 M, which was similar to the value obtained by ELISA (82 M). Thus, these data demonstrate that the reverse aYnity bead screening system is applicable to semiquantitative extraction and that compounds that speciWcally bind to a biomolecule can be puriWed and identiWed from a complex mixture in a single step. Direct puriWcation of ligand/receptor complex binding proteins Having shown that reverse aYnity beads were capable of purifying ligands from complex mixtures, we next examined whether ligand/receptor complex binding proteins could be identiWed by this approach. As is well known, ligand/receptor complex has unique structure, which may induce the recruitment of other accessory proteins to exert biological eVects. When FK506 binds to FKBP12, the FKBP12/FK506 complex associates with calcineurin A and inhibits its phosphatase activity [8–10]. When rapamycin binds to FKBP12, the FKBP12/ rapamycin complex binds to FKBP12/rapamycin-associated protein (FRAP; also known as m-TOR and RAFT1) and inhibits the serine/threonine kinase activity of FRAP [11,12]. In both examples, a ligand-speciWc structure is essential for the binding of the ligand/recep-
250
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 3. Selective puriWcation of FKBP12 binding compounds from a crude chemical mixture using reverse aYnity beads. (A) EYcient binding of FK506 to the reverse aYnity beads. The mixture of 10 compounds containing FK506 or FK506 alone (0.1 nmol) was incubated with 0.1 mg of FKBP12-immobilized beads. The eluate was quantiWed by ELISA for FK506. All data are shown as mean § SE. (B) Selective binding of FK506 to the reverse aYnity beads. The mixture of 10 compounds containing FK506 (TIC is shown at top) was incubated with 1 mg of FKBP12-immobilized beads and the TIC of the eluate is shown at bottom. (C) ConWrmation of FK506 puriWcation by LC-MS. The same data as in (B) are shown as an ion chromatogram of FK506 in (Ca). This speciWc binding was absent in the presence of a speciWc competitor, biotinylated-FK506 (Cb). (D) Selective binding of rapamycin to the reverse aYnity beads. Selective puriWcation of rapamycin was also examined from 10 compounds containing rapamycin. The eluate was analyzed by LC-MS. Da, no competitor; Db, FK506 was used as a competitor; Dc, biotinylated-FK506 was used as a competitor.
tor complex to the binding proteins [13–17] to exert biological eVects. We next reconstituted the FKBP12/FK506 complex on beads and asked whether reverse aYnity beads can purify ligand/receptor complex binding proteins in a ligand-dependent manner. FKBP12-immobilized beads were incubated with 10 g/ml FK506 for 3 h at 4 °C. The beads were then incubated with FKBP-depleted rat brain nuclear extract and binding proteins were analyzed by SDS–PAGE. When the FKBP12/FK506 complex was used for reverse aYnity puriWcation, a complex-speciWc binding protein of molecular weight 60 kDa was puriWed (Fig. 5A, lane 5, indicated by asterisk). MS-MS analysis of trypsin-digested fragments of the binding protein conWrmed it to be calcineurin A (data not shown). This was also conWrmed by immunoblotting with anticalcineurin A (Fig. 5A, below). Immunoblot analysis suggested that approximately 20% of calcineurin A in the crude extract was puriWed from the reverse aYnity beads. In contrast, calcineurin A could not be puriWed by FKBP12-immobilized beads in the absence of FK506 (Fig. 5A, lane 4).
FKBP12-immobilized beads were incubated with rapamycin to reconstitute the FKBP12/rapamycin complex. After washing, the beads were incubated with a FKBP-depleted SH-SY5Y cytoplasmic fraction. Using this approach, a protein with a molecular weight of 270 kDa was detected as a speciWc binding protein for the reconstituted FKBP12/rapamycin complex (Fig. 5B, lane 2). MS-MS analysis of tryptic-digested fragments derived from the 270-kDa binding protein conWrmed it to be FRAP (data not shown). Immunoblot analysis also conWrmed that the 270-kDa binding protein was FRAP. Furthermore, calcineurin A was also detected in this extract using FKBP12/FK506 complex (Fig. 5B, lane 3). Taken together, the results shown above indicate that our reverse aYnity bead approach eYciently puriWes ligands and interacting proteins from complex mixtures in a single step. The relatively small amount of beads required for each experiment (»20 mg of beads/assay) suggests that it will be readily possible to adapt this method for high-throughput screening. After immobilization of the biomolecules of interest to the beads, they can be put in a multiwell plate, and fermentation broth,
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
Fig. 4. One-step puriWcation of FK506-like compounds from cultured broth using reverse aYnity beads. (A) Various concentrations of broth extract from a FK506-producing strain (Streptomyces tsukubaensis) were reacted with FKBP12-immobilized beads (0.1 mg) in the presence of biotinylated FK506 (1 pmol). Bound biotinylated FK506 was detected using streptavidin–HRP. (B–E) HPLC analysis of the eluate from FKBP12-immobilized beads (20 mg) reacted with the cultured broth extract of the FK506-producing strain (0.4 ml). (B) Original broth extract; (C) eluate from control beads reacted with the broth extract; (D,E) eluate from FKBP12-immobilized beads reacted with the broth extract without D or with E biotinylated FK506.
a sponge extract, or chemical mixtures can be screened. Although there are several technologies that can monitor interaction between biomolecules and chemicals such as surface plasmon resonance and Xuorescence anisotropy, they are mainly designed only for analysis. As the beads developed in this study can recover the binding chemicals, they can be applied not only for analytical scales but also for preparative scales. In addition, reverse aYnity beads can identify ligand-dependent complex binding proteins, which is essential for drug development. This indicates that reverse aYnity beads can serve as a powerful tool for the screening of ligands, functional analysis of receptors, and signal transduction processes, all of which are important components for the development of new drugs. Based on this study, further evaluation of the beads in kinetics using other chemicals
251
Fig. 5. Direct puriWcation of ligand/receptor complex binding proteins using FKBP12/drug complex reverse aYnity beads. (A) PuriWcation of ligand-dependent binding protein using FKBP12/FK506 reverse aYnity beads. FKBP12/FK506 complex was reconstituted on FKBP12immobilized beads and incubated with rat brain nuclear extract and the eluate was analyzed by SDS–PAGE (lane 5) followed by silver staining (top) and immunoblot (bottom) analysis. Lane 1, 50 ng (for silver staining) or 20 ng (for immunoblotting) recombinant bovine calcineurin A; lane 2, control beads; lane 3, control beads preincubated with FK506; lane 4, FKBP12-immobilized beads; lane 5, FKBP12immobilized beads preincubated with FK506. An asterisk indicates the 60-kDa binding protein speciWcally bound to FKBP12/FK506 complex (Cal-A). (B) DiVerent ligands can purify distinct binding proteins using complex-reconstituted reverse aYnity beads. FKBP12/rapamycin complex and FKBP12/FK506 complex were reconstituted on FKBP12-immobilized beads and incubated with cytoplasm fraction of SH-SY5Y cells and the eluates were analyzed by SDS–PAGE. Lane 1, FKBP12-immobilized beads; lane 2, FKBP12-immobilized beads + rapamycin; lane 3, FKBP12-immobilized beads + FK506. Asterisks indicate speciWc binding proteins.
and proteins may be important to grasp the capability of the beads. IdentiWcation of ligands that act on receptors is one of the intriguing issues for drug development and in this study we showed that the “high-performance aYnity beads” can be used for ligand identiWcation and puriWcation. Since the development of aYnity puriWcation several decades ago, biochemists have puriWed only proteins because of technical limitations. Our study indicates that this technology can be reversed and shows that ligands can be puriWed by the “reverse” aYnity puriWcation.
Acknowledgments We are grateful to Fujisawa Pharmaceutical Co., Ltd. for providing FK506, rapamycin, cyclosporin A, FK506 ELISA system, and rat brain lysates and to M. Matsuura
252
Reverse aYnity beads / Y. Ohtsu et al. / Anal. Biochem. 338 (2005) 245–252
and N. Shigematsu, Fujisawa Pharmaceutical Co., Ltd., for HPLC and LC-MS analysis. We are also grateful to Y. Tsuboi and to J. Kato. We thank Professor Peter K. Vogt, Scripps Research Institute for helpful discussions. We also thank Dr. Bruce Blumberg, Department of Developmental and Cell Biology, University of California at Irvine for a critical reading of the manuscript. This work was supported by a grant of R and D Projects in Cooperation with Academic Institutions from New Energy and Industrial Technology Development Organization (NEDO). R.O. is a NEDO Fellow.
References [1] L.S. Lerman, Antibody chromatography on an immunologically speciWc adsorbent, Proc. Natl. Acad. Sci. USA 39 (1953) 232– 236. [2] N. Shimizu, et al., High-performance aYnity beads for identifying drug receptors, Nat. Biotechnol. 18 (2000) 877–881. [3] T. Tomohiro, et al., Total analysis and puriWcation of cellular proteins binding to cisplatin-damaged DNA using submicron beads, Bioconjug. Chem. 13 (2002) 163–166. [4] T. Akashi, T. Nefuji, M. Yoshida, J. Hosoda, Quantitative determination of tautomeric FK506 by reversed-phase liquid chromatography, J. Pharm. Biomed. Anal. 14 (1996) 339–346. [5] J.D. Dignam, R.M. Lebovitz, R.G. Roeder, Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res. 11 (1983) 1475–1489.
[6] F.J. Dumont, FK506, an immunosuppressant targeting calcineurin function, Curr. Med. Chem. 7 (2000) 731–748. [7] W.H. Flanagan, B. Corthesy, R.J. Bram, G.R. Crabtree, Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A, Nature 352 (1991) 803–807. [8] S.L. Schreiber, Chemistry and biology of the immunophilins and their immunosuppressive ligands, Science 251 (1991) 283–287. [9] J. Liu, et al., Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes, Cell 66 (1991) 807–815. [10] N.A. Clipstone, G.R. Crabtree, IdentiWcation of calcineurin as a key signalling enzyme in T-lymphocyte activation, Nature 357 (1992) 695–697. [11] K.E. Hentges, et al., FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse, Proc. Natl. Acad. Sci. USA 98 (2001) 13796–13801. [12] A-C. Gingras, B. Raught, N. Sonenberg, Regulation of translation initiation by FRAP/mTOR, Genes Dev. 15 (2001) 807–826. [13] J.P. GriYth, et al., X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex, Cell 82 (1995) 507–522. [14] C.R. Kissinger, et al., Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex, Nature 378 (1995) 641–644. [15] J. Choi, J. Chen, S.L. Schreiber, J. Clardy, Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP, Science 273 (1996) 239–241. [16] J. Liang, J. Choi, J. Clardy, ReWned structure of the FKBP12rapamycin-FRB ternary complex at 2.2 Å resolution, Acta. Crystallogr. D 55 (1999) 736–744. [17] G.R. Crabtree, S.L. Schreiber, Three-part inventions: intracellular signaling and induced proximity, Trends Biochem. Sci. 21 (1996) 418–422.