Identification of Small Molecule Binding Molecules by Affinity

Nov 26, 2008 - We investigated the application of resins used in solid-phase synthesis for affinity purification. A synthetic ligand for FK506-binding...
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Bioconjugate Chem. 2008, 19, 2417–2426

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Identification of Small Molecule Binding Molecules by Affinity Purification Using a Specific Ligand Immobilized on PEGA Resin Kouji Kuramochi,†,| Yuka Miyano,†,| Yoshihiro Enomoto,†,| Ryo Takeuchi,† Kazutomo Ishi,‡ Yoichi Takakusagi,† Takeki Saitoh,† Keishi Fukudome,† Daisuke Manita,† Yoshifumi Takeda,‡ Susumu Kobayashi,‡,§ Kengo Sakaguchi,†,‡ and Fumio Sugawara*,†,‡ Department of Applied Biological Science, Genome and Drug Research Center, and Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Received July 2, 2008; Revised Manuscript Received October 26, 2008

We investigated the application of resins used in solid-phase synthesis for affinity purification. A synthetic ligand for FK506-binding protein 12 (SLF) was immobilized on various resins, and the binding assays between the SLF-immobilized resins and FK506-binding protein 12 (FKBP12) were performed. Of the resins tested in this study, PEGA resin was the most effective for isolating FKBP12. This matrix enabled the isolation of FKBP12 from a cell lysate, and the identification of SLF-binding peptides from a phage cDNA library. We confirmed the interaction between SLF and these peptides using a cuvette type quartz crystal microbalance (QCM) apparatus. Our study suggests that PEGA resin has great potential as a tool not only for the purification and identification of small-molecule binding proteins but also for the selection of peptides that recognize target molecules.

INTRODUCTION To elucidate the mechanism of action of biologically active small molecules, it is important to identify their target proteins (1). Affinity purification with ligand-immobilized beads has greatly contributed to screening for small molecule target proteins (2). Generally, agarose is used as a matrix for affinity purification. Unfortunately, however, agarose has a tendency to cause nonspecific binding of proteins, which can lead to problems during the screening procedure. Moreover, the yield as well as the variety of small ligands immobilized on an agarose matrix are limited because agarose is unstable in organic solvents. Several kinds of matrices, including a glycidyl methacrylate (GMA)-covered GMA-styrene copolymer (3-7), poly(methacrylate) polymers (8-11), and poly(acrylamide) polymers (12-16), have been developed to circumvent these problems (2). We are interested in applying resins used in solidphase synthesis to the affinity purification of proteins (17). The resins have several attractive features as potential affinity matrices, including significant stability in both organic and aqueous media, chemical and physical stability, and high capacity loading of ligand. We have previously reported the affinity purification of a mammalian DNA polymerase β using its inhibitor-immobilized TentaGel resin. In earlier studies, the fluorescent, radioisotope, or biotinlabeled derivatives immobilized on affinity beads were used as tools for identification of the target proteins derived from cell lysates (18-21). Indeed, several target proteins of small molecules, such as methotrexate (7, 22, 23), FK506 (1, 5, 8-11, 24), and cyclosporin A (25-27), were isolated by these methods. Recently a phage-based method was reported (28). Phage particles can display a huge combinatorial library encoding peptides or proteins, which are derived from foreign DNA inserts * Corresponding author. Tel:+81-4-7124-1501(ext. 3400). Fax: +814-7123-9757. E-mail: [email protected]. † Department of Applied Biological Science. ‡ Genome and Drug Research Center. § Faculty of Pharmaceutical Sciences. | These authors equally contributed to this work.

Figure 1. Structure of FK506 (1) and AP1497 (2). AP1497 is one of the synthetic ligands for FKBP12 (SLFs).

as fusions of the phage capsid. Phage clones that specifically bind to the target molecules are first selected from the library. The corresponding amino acid sequences expressed on the phage surface can then be identified by sequencing the insert DNA of the phage clones. This method allows the rapid selection and identification of the ligand-binding peptides or proteins and has been successfully applied to the determination of binding proteins for FK506 (29), doxorubicin (30), HBC (31), and paclitaxel (32-34). Moreover, phage display technology can be used to efficiently identify the specific drug-binding site of the identified protein (33, 35-37). To evaluate the suitability of resins used in organic synthesis as affinity matrices, we focused on synthetic ligands for FK506binding protein 12 (SLFs) as a model system (Figure 1). AP1497 (2) is one such SLF, which tightly binds to FK506 binding protein 12 (FKBP12) with a dissociation constant (KD) of 20 nM (Figure 1) (38, 39). Previous studies demonstrated that FK506-immobilized matrices captured FKBP12 from cell lysates (1, 5, 8-11, 24) and that a biotinylated SLF derivative enabled the isolation of clones encoding FKBP12 from a T7 cDNA phage-display library (40, 41). Thus, SLF is considered an ideal model ligand to evaluate matrices for affinity purification of target proteins from both cell lysates and phage cDNA libraries. In this article, we have evaluated several resins used for organic syntheses as candidate affinity matrices for the identi-

10.1021/bc8002716 CCC: $40.75  2008 American Chemical Society Published on Web 11/26/2008

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fication of small molecule-binding proteins. We isolated FKBP12 protein from Jurkat cell lysates using SLF-immobilized PEGA resin. We also isolated two SLF-binding phage clones, which displayed full-length FKBP12 and a SLF-binding peptide, from a phage cDNA library using SLF-immobilized PEGA resin.

EXPERIMENTAL PROCEDURES General Chemistry. Dichloromethane was distilled from P2O5 prior to use. N,N-Dimethylformamide was distilled from calcium hydride prior to use. All other reagents were commercially available and used without further purification. All reactions were monitored by TLC, which was carried out on Silica Gel 60 F254 plates (Merck, Darmstadt, Germany). Flash chromatography separations were performed on PSQ 100B (Fuji Silysia Co., Ltd., Japan). 1H and 13C NMR spectra were recorded on Avance DRX-600 or DRX-400 (Bruker BioSpin, Rheinstetten, Germany), using CDCl3 (with TMS for 1H NMR and chloroform-d for 13C NMR as the internal reference) solution, unless otherwise noted. Chemical shifts were expressed in δ (ppm) relative to Me4Si or residual solvent resonance, and coupling constants (J) were expressed in Hz. Optical rotations were recorded on a P-1030 digital polarimeter (JASCO, Tokyo, Japan) at room temperature. Infrared spectra (IR) were recorded on a FT/IR-410 spectrometer (JASCO) using NaCl (neat) or KBr pellets (solid) and were reported on wavenumbers (cm-1). Mass spectra (MS) were obtained on an ABIQSTAR pulsar i mass spectrometer (Applied Biosystems, Foster City, CA) under high resolution conditions, using poly (ethylene glycol) as internal standard. Amino HypoGel200 (loading, 0.92 µmol/mg; size, 90 µm) and amino HypoGel400 (loading, 0.70 µmol/mg; size, 90 µm) were purchased from RAPP plolymere (Tu¨bingen, Germany). Amino NovaSyn resins (loading, 0.29 µmol/mg; size, 90 µm), amino NovaGel (loading, 0.83 µmol/mg; size, 75-150 µm), amino PEGA (loading, 0.40 µmol/mg; size, 150-300 µm), amino polystyrene (loading, 0.90 µmol/mg; size, 75-150 µm), and hydroxyamino Wang resins (loading, 2.0 µmol/mg; size, 100-200 µm) were purchased from novabiochem (San Diego, CA). AffiGel 102 Gels (loading, 15 µmol/mL; size, 70-300 µm) were purchased from Bio-Rad Laboratories (Hercules, CA). EAH Sepharose 4B (loading, 7-12 µmol/mL; size, 45-165 µm) was purchased from GE Healthcare Bio-Science Corp (Piscataway, NJ). The peptide (LSCLWAELSGKLWPLPAAQ) was purchased from GENIX TALK (Osaka, Japan). General Biology. Fetal bovine serum (FBS) was purchased from Biowest (Loire Valley, French). Kanamycin sulfate, dimethyl sulfoxide (DMSO), and biotin were purchased from Wako Pure Chemical Industries (Tokyo, Japan). RPMI 1640 and 2-Mercaptoethanol were purchased from Nacalai Tesque (Kyoto, Japan). Protease inhibitor cocktail was obtained from Sigma-Aldrich (ST. Louis, MO). Streptavidin agarose gel was purchased from GE Healthcare (Buckinghamshire, U.K.). (1R)-1-[3-[2-(4-Nitrophenoxy)-2-oxoethyl]phenyl]-3-(3,4dimethoxyphenyl)-1-propanyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-piperidinecarboxylate (3). Compound 3 was prepared in ca. 4:1 tautomeric mixture according to our previous report (41). Major tautomer: [R]D24 ) -7.2 (c 1.8, CHCl3); 1H NMR (600 MHz, CDCl3) δ 8.27 (2H, m), 7.33 (2H, m), 7.31 (1H, m), 7.03 (1H, d, J ) 7.7 Hz), 7.00 (1H, brs), 6.93 (1H, dd, J ) 8.2 Hz, 2.5 Hz), 6.77 (1H, m), 6.68 (2H, m), 5.80 (1H, dd, J ) 7.9 Hz, 5.7 Hz), 5.32 (1H, brd, J ) 5.3 Hz), 4.96 (2H, s), 3.85 (6H, s), 3.35 (1H, brd, J ) 12.7 Hz), 3.16 (1H, td, J ) 13.1 Hz, 2.9 Hz), 2.57 (2H, m), 2.36 (1H, m), 2.24 (1H, m), 2.07 (1H, m), 1.72 (5H, m), 1.45 (1H, m), 1.35 (2H, m), 1.22 (3H, s), 1.21 (3H, s), 0.88 (3H, t, J ) 7.4 Hz); 13C NMR (100 MHz, CDCl3) δ 207.8, 169.7, 167.3, 166.6, 157.7, 154.6, 148.9, 147.4, 145.6, 141.8, 133.3, 129.9, 125.3 (× 2), 122.2 (× 2), 120.4, 120.1, 114.6, 113.1, 111.7, 111.3, 76.4, 65.3, 55.9, 55.8,

Kuramochi et al.

51.2, 46.7, 44.1, 38.0, 32.4, 31.2, 26.3, 24.9, 23.4, 23.1, 21.1, 8.7; IR (neat) 3021, 2937, 2861, 1786, 1738, 1701, 1640, 1592, 1521, 1444, 1348, 1210, 1143, 1082, 1029, 992, 921, 864, 756 cm-1; HRMS (ESI) calcd for C38H44N2O11 ([M + Na]+) m/z 727.2837, found 727.2800. General Method for the Preparation of SLF-Immobilized Resins (5). Dry resins (1 equiv) were swollen in pyridine. To the suspension of the resins was added a solution of 3 (0.3-1.9 equiv) and DMAP (1.2 equiv) in CH2Cl2. After the mixture was stirred at r.t. for 19 h, the mixture was filtrated and washed with CH2Cl2 five times. The filtrate was concentrated, and the residue was purified by silica gel chromatography (hexane/ EtOAc ) 4:1 to 1:1) to give the recovered AP1497 p-nitrophenyl ester. The amount of SLF immobilized on the dry resins was estimated by the amount of the recovered 3. The SLF-immobilized resins were suspended in Ac2Opyridine (1:1), and the suspension was stirred at r.t. for 3 h. Then the suspension was filtered and washed with CH2Cl2 three times, MeOH three times, and CH2Cl2 twice. The resulting resins were dried in vacuo. The amount of SLF immobilized on 1 mg of dry PEGA resin (5e) was 0.19 µmol. Because the swelling volume of PEGA in water is reported to be 16 mL/g in the manufacturer’s instruction, 1 g of PEGA resin swells up to 16 mL. Thus, the amount of SLF immobilized on 1 mL of wet PEGA resin (5e) was estimated to be 11-12 µmol. General Method for the Preparation of Acetylated Resins (Control Resins) (6). Dry resins were swollen in pyridine (5.0 mL). To the suspension of the resins was added Ac2O (5.0 mL), and the suspension was stirred at r.t. for 3 h. Then the suspension was filtered and washed with CH2Cl2 three times, MeOH three times, and CH2Cl2 twice. The resulting resins were dried in vacuo. Synthesis of SLF-Immobilized AffiGel. Affigel 102 Gel was washed with DMF five times and swollen in DMF (5). To the suspension of the gel was added a solution of 3 (1.2 equiv) and DMAP (1.2 equiv) in DMF. After the mixture was stirred at r.t. for 16 h, the mixture was filtrated and washed with DMF five times. The filtrate was concentrated, and the residue was purified by silica gel chromatography (hexane/EtOAc ) 4:1 to 1:1) to give the recovered 3. The amount of SLF immobilized on the gel was estimated to be 15 µmol/mL by the amount of recovered 3. The SLF-immobilized AffiGel were suspended in 20% DMF solution of Ac2O, and the suspension was stirred at r.t. for 3 h. Then the suspension was filtered and washed with DMF three times and 20% aqueous solution of EtOH three times. Synthesis of Acetylated AffiGel. Affigel 102 Gel was washed with DMF five times. The gel was suspended in 20% DMF solution of Ac2O, and the suspension was stirred at r.t. for 3 h. Then the suspension was filtered and washed with DMF three times and 20% aqueous solution of EtOH three times. Synthesis of SLF-Immobilized Sepharose 4B. EAH Sepharose 4B was washed with 1 mM aqueous solution of HCl three times and in 0.5 M aqueous NaCl, and swollen in dioxane/H2O (1:1). To the suspension of the resins was added a solution of 3 (1.2 equiv) and DMAP (1.2 equiv) in dioxane/H2O (1:1). After the mixture was stirred at r.t. for 16 h, the mixture was filtrated and washed with dioxane/H2O (1:1) five times. The filtrate was concentrated, and the residue was purified by silica gel chromatography (hexane/EtOAc ) 4:1 to 1:1) to give recovered 3. The amount of SLF immobilized on the beads was estimated to be 7 µmol/mL by the amount of recovered 3. The SLFimmobilized Sepharose 4B were suspended in 5% Ac2O in dioxane, and the suspension was stirred at r.t. for 3 h. Then the suspension was filtered and washed with dioxane/H2O five times and H2O three times.

PEGA Resin As an Affinity Matrix

Synthesis of Acetylated Sepharose 4B. EAH Sepharose 4B was washed with 1 mM aqueous solution of HCl three times and in 0.5 M aqueous NaCl, and swollen in dioxane/H2O (1:1). The gels were treated with 5% Ac2O in dioxane, and the suspension was stirred at r.t. for 3 h. Then the suspension was filtered and washed with dioxane/H2O five times and H2O three times. Bis(SLF-PEG-undecyl)disulfide (7). Compound 7 was prepared in ca. 4:1 tautomeric mixture from compound 3 according to our previous report (41). Major tautomer: [R]D24 ) -3.6 (c 0.67, CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.31 (1H × 2, t, J ) 7.9 Hz), 7.05 (1H × 2, brt, J ) 5.6 Hz), 7.00 (1H × 2, brd, J ) 7.7 Hz), 6.93 (1H × 2, brs), 6.86 (1H × 2, dd, J ) 7.9 Hz, 2.0 Hz), 6.78 (1H × 2, m), 6.68 (1H × 2, brs), 6.67 (2H × 2, m), 6.09 (1H × 2, brm), 5.77 (1H, dd, J ) 7.9 Hz, 5.6 Hz), 5.31 (1H × 2, d, J ) 5.4 Hz), 4.50 (2H × 2, s), 3.86 (3H × 2, s), 3.85 (3H × 2, s), 3.63 (8H × 2, brs), 3.60 (2H × 2, m), 3.57 (2H × 2, m), 3.52 (2H × 2, m), 3.44 (2H × 2, m), 3.37 (1H × 2, brd, J ) 12.3 Hz), 3.18 (1H × 2, td, J ) 13.1 Hz, 2.9 Hz), 2.67 (2H × 2, t, J ) 7.4 Hz), 2.61 (1H × 2, m), 2.53 (1H × 2, m), 2.38 (1H × 2, brd, J ) 13.7 Hz), 2.25 (1H × 2, m), 2.15 (2H × 2, t, J ) 7.4 Hz), 2.05 (1H × 2, m), 1.80-1.60 (12H × 2, m), 1.50 (1H × 2, m), 1.35 (4H × 2, m), 1.27 (10H × 2, brs), 1.23 (3H × 2, s), 1.21 (3H, s), 0.89 (3H × 2, t, J ) 7.4 Hz); 13C NMR (100 MHz, CDCl3) δ 207.8 (× 2), 173.2 (× 2), 169.7 (× 2), 168.1 (× 2), 167.3 (× 2), 157.4 (× 2), 148.9 (× 2), 147.4 (× 2), 141.9 (× 2), 133.3 (× 2), 130.0 (× 2), 120.1 (× 2), 120.1 (× 2), 114.2 (× 2), 113.3 (× 2), 111.3 (× 2), 76.5 (× 2), 70.5 (× 4), 70.3 (× 2), 70.2 (× 2), 70.0 (× 2), 69.7 (× 2), 67.4 (× 2), 55.9 (× 2), 55.8 (× 2), 55.8 (× 2), 51.3 (× 2), 46.7 (× 2), 44.1 (× 2), 39.12 (× 2), 39.10 (× 2), 38.8 (× 2), 36.7 (× 2), 32.5 (× 2), 31.2 (× 2), 29.7 (× 2), 29.5 (× 2), 29.42 (× 2), 29.36 (× 2), 29.3 (× 2), 29.21 (× 2), 29.20 (× 2), 28.5 (× 2), 26.4 (× 2), 25.7 (× 2), 24.5 (× 2), 23.5 (× 2), 23.2 (× 2), 21.2 (× 2), 8.7 (× 2); IR (neat) 3342, 2928, 2857, 1738, 1645, 1517, 1444, 1351, 1261, 1141, 1030, 992 cm-1; HRMS (ESI) calcd for C102H56N6O24Na2S2 979.5198 ([M + 2Na]2+), found 979.5220. Calcd for C102H56N6O24NaS2 m/z 1936.0504 ([M + Na]+), found 1936.0549. Bis(Acetyl-PEG-undecyl)disulfide (Bis 11-(N-acetyl-3,6,9trioxaundecyl-N-carbamoylundecyl)disulfide) (8). A solution of bis(amino-PEG-undecyl)disulfide (13.4 mg, 13.3 µmol) in Ac2O (1 mL) and pyridine (1 mL) was stirred at r.t. for 20 h. Then the mixture was concentrated, and the residue was purified by silica gel chromatography (CHCl3/MeOH ) 20:1) to give 7 (10.4 mg, 90%) as a white solid. Mp ) 88-89 °C; 1H NMR (600 MHz, CDCl3) δ 6.22 (1H ×2, brs), 6.11 (1H × 2, brs), 3.65 (8H × 2, brs), 3.57 (4H × 2, t, J ) 5.1 Hz), 3.45 (4H × 2, m), 2.68 (2H × 2, t, J ) 7.3 Hz), 2.17 (2H, x 2, t, J ) 7.9 Hz), 1.99 (3H × 2, s), 1.69-1.60 (4H × 2, m), 1.37 (2H × 2, m), 1.27 (10H × 2, brs); 13C NMR (100 MHz, CDCl3) δ 173.2 (× 2), 170.0 (× 2), 70.39 (× 2), 70.38 (× 2), 70.1 (× 4), 69.9 (× 2), 69.8 (× 2), 39.2 (× 2), 39.1 (× 2), 39.0 (× 2), 36.6 (× 2), 29.36 (× 2), 29.33 (× 2), 29.27 (× 2), 29.2 (× 2), 29.1 (× 4), 28.4 (× 2), 25.6 (× 2), 23.1 (× 2); IR (neat) 3298, 3082, 2921, 2849, 1643, 1557, 1463, 1414, 1377, 1286, 1232, 1143, 997, 952 cm-1; HRMS (ESI) calcd for C42H82N4O10NaS2 889.5364 ([M + Na]+), found 889.5364. Expression of Six Histidine-Tagged FK506-Binding Protein 12 (FKBP12). The cDNA encoding human FKBP12 was cloned into the expression vector pET28a(+) (Novagen) and was transformed into Esherichia coli BL21(DE3). A single colony was inoculated into 15 mL of LB medium containing 1% glucose and 50 µg/mL kanamycin, and cultured at 30 °C. The cells were grown overnight and then were transferred into 1 L of the same medium at 37 °C. After incubation for 3 h, the expression of FKBP12 was induced by adding IPTG to 1 mM. The culture was then continued for further 3 h at 37 °C. The

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cultured cells were harvested by centrifugation at 1500g for 10 min at 4 °C. The cells were resuspended in PBS and centrifuged at 1100g for 10 min at 4 °C. After removal of the supernatant, the cells were frozen using liquid N2 and then stored at -80 °C. Purification of His-Tagged FKBP12. The FKBP12-producing cells were sonicated in buffer A (50 mM phosphate (pH. 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL leupeptin, and 1 µg/mL pepstatin A) and then centrifuged at 20400g for 30 min at 4 °C. The supernatant was filtered through a PVDF membrane (Millex, pore size 0.45 µm, Millipore, Billerica, MA) and then loaded onto a Ni-NTA resin (GE Healthcare) pre-equilibrated with buffer A. The column was washed with buffer B (50 mM phosphate (pH. 8.0), 300 mM NaCl, 20 mM imidazole), and buffer C (50 mM phosphate (pH. 8.0), 300 mM NaCl, and 30 mM imidazole). Bound proteins were then eluted with buffer D (50 mM phosphate (pH. 8.0), 300 mM NaCl, and 250 mM imidazole). The fractions containing the His-tagged FKBP12 were collected and dialyzed into TEMG buffer (50 mM TrisHCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 10% glycerol, and 5 mM 2-mercaptoethanol) for purification by FPLC. The protein was loaded onto HiTrap DEAE column pre-equilibrated with TEMG buffer, and the flow-through fraction was then subjected to HiTrap SP column. The flow-through fraction was collected and dialyzed into FKBP buffer (25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 5 mM 2-mercaptoethanol, and 0.05% Nonidet P-40) and then stored at -80 °C until use. Binding Analysis between FKBP12 and the Resins. Each resin (4 mg) was pre-equilibrated with the FKBP12 buffer (400 µL) overnight. Then 600 µL of recombinant FKBP12 (1.61 mg/ mL, 0.97 mg) was added to the resins. The resins were incubated at 4 °C for 4 h. The amount of FKBP12 in the supernatant was determined by the Bradford assay, with absorbance measured at 595 nm. The amount of FKBP12 bound to the resins was calculated by subtracting the amount of FKBP12 in the supernatant from the amount of total FKBP12. After the resins were washed with the FKBP12 buffer (850 µL) 5 times, the resulting resins were boiled at 90 °C in 250 µL of SDS-PAGE loading buffer (0.25 mM dithiothreitol, 0.5% SDS, 20 mM TrisHCl (pH6.8), 2.5% glycerol, and 0.01% Bromo phenol blue). The supernatant of each sample was separated on a 15% acrylamide gel, visualized by CBB staining, and quantified using the ImageJ program. Affinity Purification of FKBP12 from Jurkat Cell Lysates by the SLF-Immobilized PEGA Resin. All procedures were performed at 4 °C. Frozen Jurkat cells (1 × 107 cells) were suspended in 1 mL FKBP buffer containing protease inhibitors (1 mM phenylmethyl sulfonyl fluoride, 1 µg/mL leupeptin, and 1 µg/mL pepstatin A), sonicated twice for 5 s, and incubated on ice for 25 min. The cells were centrifuged at 15000 rpm for 20 min at 4 °C, and the supernatant was kept as cell lysate. After SLF-immobilized PEGA resin (4 mg) and control PEGA resin (4 mg), pre-equilibrated with FKBP buffer containing protease inhibitors, the resulting beads were added to 450 µL of Jurkat cell lysate. The mixtures (1 mL) were rotated gently for 4 h. The resins were washed 3 times with 850 µL of FKBP buffer containing protease inhibitors and were boiled at 90 °C in 50 µL of SDS-PAGE loading buffer (1.25 mM dithiothreitol, 2.5% SDS, 100 mM Tris-HCl (pH6.8) 12.5% glycerol, and 0.01% bromo phenol blue). Each sample was separated on a 15% acrylamide gel and visualized by silver staining. FKBP12 bound to the resins was detected by Western blotting with anti-FKBP12 goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and alkali phosphataseconjugated antigoat IgG.

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Construction of FKBP12 Phage Clone. Full length gene encoding human FKBP12 protein was obtained from a phage cDNA library by PCR with the two primers, FKBP-up (5′GAATTCGATGGGAGTGCAGGTGGAAAC-3′) and FKBPdown (5′-AAGCTTTCATTCCAGTTTTAGAAGCTCC-3′). The amplified DNA was double digested with EcoR I and Hind III endonucleases and concentrated by ethanol precipitation. The purified DNA was then ligated into T7-Select10-3b vector (Novagen) and subsequently packaged into phagemid (Novagen). The FKBP12 phage clone was amplified by infection into E. coli strain BLT5615. Binding Analysis of FKBP12 Phage Clone to the SLF-Immobilized Resin or Control Resin. Each resin (100 µg) was pre-equilibrated with TBS (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% Glycerol, and 0.05% Nonidet P40) for 12 h at 4 °C. The FKBP12 phage clone was amplified by infection into E. coli strain BLT5615 as host cells, and the amplified phage clone (1 × 1010 plaque forming units (pfu)/mL) was added to the resin. After incubation at room temperature for 1 h, the resin was precipitated and the phage solution removed. The resin was then washed 10 times with Wash buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% Glycerol, 0.05% Nonidet P40, and 5 mM 2-mercaptoethanol). Bound phage particles were subsequently removed from the resin by 15 min incubation in 1% SDS in TBS buffer. The eluted phage particles were amplified by infection into Escherichia coli strain BLT5615, and the titers of the phage particles from each resin were calculated. The phage clone with no inserted cDNA fragment (No Ins phage) was used in the same binding assay as the negative control. Construction of T7 cDNA Phage Library. The T7 phage library was constructed using cloning kits for T7 phage display (T7 Select 10-3 OrientExpress cDNA Cloning System, Random Primer, Novagen) and cDNA derived from Colo205 cells according to the method given by the manufacturer. The primary titer of this T7 phage library was 3.3 × 105 pfu. The library was amplified with a titer of 1 × 1010 pfu/mL for screening. Affinity Selection of SLF-Binding Phage Clones from T7 cDNA Phage Library Using the SLF-Immobilized PEGA Resin. A modified version of the biopanning technique given in the manufacturer’s instructions (Novagen) was used in this study. The resin (100 µg) pre-equilibrated with TBS (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, and 0.05% Nonidet P40) for 12 h at 4 °C. The T7 cDNA display library (1 mL, approximately 1 × 1010 pfu/mL) was added to the resin. After incubation at room temperature for 1 h, the resin was precipitated and the phage solution removed. The resin was then washed 10 times with Wash buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 0.05% Nonidet P40, and 5 mM 2-mercaptoethanol). Adsorbed phage particles were subsequently removed from the resin by 15 min of incubation in 1% SDS solution. The eluted phage particles were amplified by infection into E. coli strain BLT5615 as host cells. The amplified phage was applied to subsequent rounds of biopanning. The titer was monitored at each panning step. The eluted phage solution at each step was used for the immediate infection of host cells. Incubation with shaking at 37 °C was continued until lysis was observed. Amplified phage titers of each round were estimated to be approximately 1-6 × 1010 pfu/mL. After each round, the eluted phage titers were compared to those of the previous round. Characterization of Binding Phage. After the seventh round, the eluted phage particles were applied to Luria-Bertani (LB) plates containing 50 µg/mL carbenicillin. Sixteen clones were randomly selected from the plate, and each clone was dissolved in phage extraction solution (20 mM Tris-HCl pH 8.0, 100 mM

Kuramochi et al. Scheme 1. Synthesis of SLF-Immobilized Supports (5a-j) and Control Supports (6a-j)a,b

a (a) HypoGel 200, (b) HypoGel 400, (c) TentaGel, (d) NovaSynTG, (e) PEGA, (f) NovaGel, (g) polystyrene resins, (h) Wang resins (i) AffiGel, (j) Sepharose 4B. b The loading of SLF on 5 was estimated from the amount of the recovered 2, resulting in 5a (0.48 µmol/mg), 5b (0.49 µmol/mg), 5c (0.23 µmol/mg), 5d (0.19 µmol/mg), 5e (0.19 µmol/mg), 5f (0.48 µmol/mg), 5g (0.48 µmol/mg), 5h (0.48 µmol/mg), 5i (15 µmol/mL), and 5j (7 µmol/mL).

NaCl, and 6 mM MgSO4). Phage DNA carrying insert cDNAs was amplified by PCR using a forward primer (5′-TGCTAACTTCCAAGCGGACC-3′) and a reverse primer (5′-TTGCCCAGAACTCCCCAA-3′). The PCR products were purified with ExoSAP-It (GE Healthcare) and used for cycle sequence PCR with a BigDye Terminator 3.1 Cycle Sequence Kit (Applied BioSystems). The resulting fragments were precipitated with ethanol. The purified PCR products were sequenced on an ABI PRISM 3100 Genetic Analyzer (Applied BioSystems). From the results, the deduced amino acid sequence present on the T7 phage capsid was determined. Kinetic Analysis of the Binding between SLF and FKBP12 or SLF-Binding Peptide by QCM Apparatus. Kinetic analyses were performed by a 27 MHz QCM system (Affinix Q, Initium Inc., Tokyo, Japan). Disulfide linked SLF dimer or acetylated diulfide was immobilized on the gold electrode of the sensor chip (41). The sensor chip was set on the QCM apparatus and soaked in buffer (8 mL, 10 mM TrisHCl, pH 8.0, and 200 mM NaCl). The frequency of the QCM was defined as zero position after equilibrium (3 min). Then an 8 µL aliquot of His-tagged FKBP12 or 20 amino acid peptide sequence (SLSCLWAELSGKLWPLPAAQ) was sequentially injected into the cuvette. The changes of the frequency were monitored over time. The kinetic analysis was performed by AQUA ver. 1.5 software (Initium Inc.).

RESULTS Syntheses of SLF-Immobilized Supports and Control Supports. AP1497 (2) was immobilized on resins (4a-h), which are normally used for solid-phase syntheses, and commercially available agarose matrices (4i and 4j) by two steps via its nitrophenyl ester 3 (Scheme 1) (38, 41). After immobilization of 2 onto the resins, the residual amino groups were blocked with acetic anhydride, yielding SLF-immobilized supports (5a-j). The loading of the SLF molecule was estimated from the amount of recovered 3. Typically, the SLF-immobilized supports were prepared with a loading of 0.007-0.49 µmol/ mg. In addition, control matrices (6a-j) were prepared by acetylation of 4 with Ac2O to check the binding specificity.

PEGA Resin As an Affinity Matrix

HypoGel200 (4a), Hypogel400 (4b), TentaGel (4c), and NovaSyn TG (4e) are polystyrene-polyethylene glycol graft polymers (42). The molecular masses of polyethylene glycol (PEG) in HypoGel200 and Hypogel400 are 200 and 400 Da, respectively. TentaGel and NovaSynTG resins have PEG chains with a molecular mass of about 2000-3000 Da. The major difference between TentaGel and NovaSyn TG is the structure of the linkers. The PEG chains in TentaGel are linked to the polystyrene core via benzyl ether or in the case of NovaSyn TG via ethyl ether. NovaGel (4f), (4′-O-methylpolyethyleneglycoxycarbonylaminomethyl)polystyrene, was prepared from a modified aminomethylated polystyrene by partial derivatization with PEG chains. NovaGel has PEG chains with a molecular mass of about 2000 Da. PEGA (4e) is a copolymer of dimethylacrylamide and PEG (43). The molecular mass of the PEG chains in PEGA resin is about 800 Da. Wang resin (4h) was linked to the polystyrene core via the hydroxymethylphenoxy group. AffiGel (4i) and Sepharose 4B (4j) are commercially available agarose supports and are usually used for target identification of biologically active compounds. All of the supports used in this paper had a similar particle size (45-300 µM). Binding Analyses between FKBP12 and the SLF-Immobilized Supports. Binding analyses between recombinant FKBP12 and the resins for organic synthesis were performed by the Bradford method for protein quantification (Figure 2A) (17, 44). An excess amount of FKBP12 (0.97 mg) was applied to each SLF-immobilized resin (5a-h) or control resin (6a-h). After incubation at 4 °C for 4 h, the amount of FKBP12 in the supernatant was determined using the Bradford assay. The amount of FKBP12 bound to the resins was calculated by subtraction of the amount of free FKBP from that of total FKBP12. As shown in Figure 2A, only SLF-immobilized PEGA resin (5e) bound a significant amount of FKBP12 (0.53 mg), i.e., >25-fold greater than that bound by the control PEGA resin (6e) (0.02 mg). After washing away the unbound FKBP12 with buffer 5 times, the resins were boiled with SDS sample buffer. Any eluted FKBP12 was detected by SDS-PAGE and visualized using CBB staining. Figure 2B shows that FKBP12 was eluted only from 5e. In contrast, no bound FKBP was detectable using the other resins, even after loading in the presence of abundant quantities of FKBP. The binding specificity was also confirmed by a competition assay with free SLF (Supporting Information). Similarly, we compared the amount of FKBP12 binding to SLF immobilized on PEGA resin (5e) with that to AffiGel (5i), which is one of the matrixes widely utilized for affinity purification of proteins (Figure 2C). SDS-PAGE analyses indicated that very similar amounts of FKBP12 were captured by the SLF on both resins in significantly low background. We confirmed that both 5e and 5i absorbed almost the same amount of FKBP12 (approximately 180 µg/4 mg resin). Because the amount of SLF immobilized on 5e and 5i was 11-12 and 15 µmol/mL, respectively (see Experimental Procedures and ref 45 for details), these data indicate that FKBP12 specifically bound to SLF immobilized on PEGA resin with efficiency similar to that of the AffiGel matrix. In addition, we confirmed that the PEGA resin with other ligands, such as lithocolic acid (LCA, a DNA polymerase β inhibitor) and glutathione (GSH), was applicable for affinity purification (Supporting Information). We next sought to isolate FKBP12 from Jurkat cell lysates with the SLF-immobilized PEGA resin (5e) in a small scale. After incubation with the cell lysates, the resins were washed and then bound proteins were analyzed by SDS-PAGE. A 12kDa protein was detected in the eluted proteins from 5e in the silver-stained gel but not in those from 6e (Figure 3A). In addition, a polyclonal antibody against FKBP12 cross-reacted

Bioconjugate Chem., Vol. 19, No. 12, 2008 2421

Figure 2. Binding analysis between recombinant FKBP12 and SLFimmobilized resins. (A) The amount of FKBP12 binding to resins 5a-h and 6a-h. A total of 0.97 mg of FKBP12 was applied onto 5a-h and 6a-h. After equilibrium, the amount of FKBP12 in the supernatant was determined by the Bradford assay. The amount of FKBP12 binding to resins 5a-h and 6a-h was calculated by subtraction of the amount of FKBP12 in the supernatant from the input FKBP12. (B) SDS-PAGE of FKBP eluted from 5a-h and 6e. After washing the resins to remove unbound FKBP12, the resins were boiled with SDS sample buffer. The eluted FKBP12 was analyzed by SDS-PAGE and visualized by CBB staining. Lane 1, molecular weight marker; lane 2, input FKBP12 (20%); lane 3, 5a; lane 4, 5b; lane 5, 5c; lane 6, 5d; lane 7, 5f; lane 8, 5g; lane 9, 5h; lane 10, 6e; lane 11, 5e. (C) Comparison of FKBP12 binding between SLF-immobilized PEGA resin (5e) and AffiGel (5i). The bound FKBP12 protein on each resin was separated on a SDS-polyacrylamide gel (right) and quantified using ImageJ software (left). Lane 1, input FKBP12 (100%); lanes 2-5, elution from 6e and 5e, and 6i and 5i, respectively.

with this protein band (Figure 3B), indicating that the SLFimmobilized PEGA resin efficiently captured FKBP12 from the cell lysate. Binding Analysis between FKBP12 Phage Clone and SLF-Immobilized PEGA Resin. To explore the suitability of PEGA resin as an affinity matrix for phage display, we conducted a T7 phage display screen using the SLF-immobilized PEGA resin. The binding of the T7 phage expressing FKBP12 clone (FKBP12 phage) to the SLF-immobilized PEGA resin was examined (Figure 4A). The phage particles were incubated with the SLF-immobilized PEGA (5e) or the control PEGA resins (6e). After removal of unbound phage, the bound phage particles were eluted and counted by infection into E. coli. The

2422 Bioconjugate Chem., Vol. 19, No. 12, 2008

Kuramochi et al.

Figure 3. Affinity purification and detection of FKBP12 from Jurkat cell lysates using the SLF-immobilized PEGA resin. (A) SDS-PAGE analysis of the eluted samples from SLF-immobilized PEGA resin (5e) and control PEGA resin (6e). The protein bands on the gel were visualized after silver staining. Lane 1, molecular weight marker; lane 2, cell lysate; lane 3, elution from 6e; lane 4, elution from 5e. (B) Immunoblotting analysis for FKBP12. The eluted fractions were subjected to immunoblotting analysis using antihuman FKBP12 antibody. Lane 1, cell lysate; lane 2, elution from 6e; lane 3, elution from 5e.

recovery rate of the eluted phage particles from 5e or 6e is shown in Figure 4. 5e trapped 600-fold more FKBP12 phages than 6e. In contrast, there was little difference between the recovery rates of the phage with no insert cDNA (No Ins phage) from the two resins (i.e., 5e and 6e). These results indicate that the FKBP12 phage clone specifically binds SLF immobilized on the PEGA resin. We conducted the same binding analyses using AffiGel and Sepharose 4B (Figure 4 B and 4C). The SLFimmobilized AffiGel (5i) trapped 144-fold more FKBP12 phages than the acetylated AffiGel (6i), although the recovery rate was quite lower than that in the case of 5e (Figure 4B). By contrast, the SLF-immobilized Sepharose 4B (5j) absorbed the FKBP12 phage clone with significantly high background. Our results indicate that PEGA resin may be a useful matrix for phage display screening. Affinity Selection of SLF-Binding Phage Clones from the T7 Phage cDNA Library Using the SLF-Immobilized PEGA Resin. Isolation of FKBP12 phage from a phage cDNA library using the SLF immobilized PEGA resin was examined (Figure 5A). The T7 phage cDNA library was prepared with total RNA from Colo205 cells. The primary T7 phage library contained approximately 3.3 × 105 individual phage clones. The phage cDNA library was incubated with the SLF-immobilized PEGA (5e) or control PEGA resin (6e). After removal of unbound phage particles, the bound phage particles were eluted and amplified in E. coli. The resulting phages were then used in the next round of selection. Using 5e, the recovery rate of eluted phage particles against input phage particles increased with each round of biopanning (Figure 5). By contrast, this ratio displayed little variation during each round of biopanning using 6e (Figure 5). After the seventh round, we randomly selected 13 clones out of the eluted phage clones from 5e. We then analyzed the deduced amino acid sequences displayed on the phage surface (Table 1A). Three clones (clone 1) encoded fulllength FKBP12 including the 5′-terminal untranslated region. The other 9 clones displayed 20 amino acids (SLSCLWAELSGKLWPLPAAQ) on the phage surface, which are derived from a frameshift of the inserted cDNA. We also randomly selected 15 clones from the phages binding to 6e and analyzed the corresponding sequences. Fourteen out of 15 clones binding to

Figure 4. Binding analysis between FKBP12 phage clone and SLFimmobilized PEGA resin (A), AffiGel (B), or Sepharose 4B (C). Recovery rate of FKBP12 phage particles to the input phage particles from SLF-immobilized beads (filled column) or control beads (open column) is indicated. The phage clone with no insert cDNA (No Ins phage) was used in the same binding assay as the negative control. Values are the mean ( SD (n ) 6).

6e encoded an individual short peptide, but no consensus sequences were identified. Specific binding of clone 2 onto 5e was confirmed using the same procedures as for FKBP12 phage (Figure 6). The recovery rate of clone 2 from 5e was 9.5-fold higher than that from 6e. However, there are no differences between the recovery of No Ins phage clone by 5e and 6e. These results indicate that clone 2 specifically recognizes SLF immobilized on the PEGA resin. Taken together, these results indicate that affinity selection of SLF-binding phage clones from the cDNA library was successfully achieved by the use of the SLF-immobilized PEGA resin. We searched for human genes homologous to the clone 2 peptide using PSI-BLAST (position-specific iterative BLAST). The clone 2 peptide displayed homology to various human gene products, but showed a relatively higher level of similarity to the 13 amino-acid sequence of group E Sox proteins (SoxE) (Supporting Information). This result suggests that SLF may bind to many kinds of proteins in human cells and that SoxE members are passive targets of SLF in vivo. Kinetic Analysis of the Binding between SLF and the SLF-Binding Peptide Using a QCM Apparatus. We performed a kinetic analysis of the interaction between SLF and the clone 2 peptide (SLSCLWAELSGKLWPLPAAQ) using a cuvette type quartz crystal microbalance (QCM) apparatus (Affinix Q, Initium). Disulfide linked with SLF (Bis(SLF-PEGundecyl)disulfide; 7) or acetylated disulfide (bis(Acetyl-PEGundecyl)disulfide; 8) was immobilized onto the gold surface of

PEGA Resin As an Affinity Matrix

Bioconjugate Chem., Vol. 19, No. 12, 2008 2423

Figure 6. Binding analysis between the clone 2 and SLF-immobilized PEGA resin. Recovery rate of clone 2 to the input phage particles obtained from SLF-immobilized PEGA resin (filled column) or control PEGA resin (open column) is indicated. The phage clone with no insert cDNA (No Ins phage) was used in the same binding assay as the negative control. Values are the mean ( S.D. (n ) 6).

Figure 5. Recovery rate of SLF-binding phage particles after each round of biopanning. (A) The recovery rate obtained from the SLFimmobilized PEGA resin (5e). (B) The recovery rate obtained from the control PEGA resin (6e). The phage cDNA library derived from Colo205 cells was applied to 5e or 6e. After removal of unbound phage particles, bound phage particles were eluted and amplified by infection of E. coli. The resulting phage particles were used in the next round of selection. At each round, the recovery rates of the eluted phage particles to the input phage particles from the resins were calculated. Table 1. Amino Acid Sequence of Peptide Displayed on the Phage Particle after the Biopanning Using (A) SLF-Immobilized PEGA Resin (5e) or (B) Control PEGA Resin (6e) (A) phage clone

sequencea

frequencyb

1c

2

GRCWSTPPVAPPARSASAAA MGVQVETISPGDGRTFPKRG QTCVVHYTGMLEDGKKFDS SRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISP DYAYGATGHPGIIPPHATLV FDVELLKLE SLSCLWAELSGKLWPLPAAQ

phage clone

sequencea

frequencyb

PAGISRELVDKLAAALE SHVRESGARTKAAVAQ RNLLWLE SGASPAV SPTLTHF TEKRQEDPYYCPRDR SAVCTKGRDLINASL SGTTVQSGGLGQGPGGLAKAS PVAGGPLGREVR SHSSGLNDHSHHGPGARHLPP PQGHAAWPP SSKHDF SPSRSRTLGSPAGGSGPGQAG REGEGGPGAAGQTGHR SVRGRKNIPQPSTLKQWGEGS QRVQMAPVPFAAEN SQEVSEKLPQG SKTIASSSPPS

2/15 1/15 1/15 1/15 1/15 1/15 1/15 1/15

3/13

9/13

(B) 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′

Figure 7. Structure of the disulfide linked with SLF (7) and acetylated disulfide (8).

the sensor chip (Figure 7). After washing the gold surface, the resulting sensor chip was mounted onto the QCM apparatus. As a positive control, recombinant FKBP12 was injected into the cuvette, and the QCM frequency shifts caused by the binding between the protein and small molecules conjugated to the sensor chip were monitored (red line in Figure 8A). The injection of protein significantly varied the frequency on the SLF-immobilized sensor chip. However, these frequency changes were only slight on the compound 8-immobilized sensor chip, which was used as a negative control (Figure 8A, black line). Similarly, the addition of clone 2 peptide caused substantial frequency changes on the SLF-immobilized sensor chip but almost no change on the compound 8-immbilized sensor chip (Figure 8B). The binding of FKBP12 or the clone 2 peptide against compound 7 was analyzed using the Langmuir adsorption model (Figure 9). The KD values of the recombinant FKBP12 and clone 2 peptide with compound 7 were 0.027 µM and 5.8 µM, respectively. These results indicate that the clone 2 peptide (SLSCLWAELSGKLWPLPAAQ) specifically binds to SLF in vitro.

1/15 1/15 1/15 1/15 1/15 1/15

a Amino acid sequences were determined from DNA sequences of the insert DNA. b Frequency is the number of sequences among the total phage clones selected. c The underlined sequence corresponds to the full-length sequence of the human FKBP12 protein.

DISCUSSION In this study, we investigated whether the resins used for solid-phase syntheses were suitable for affinity purification. To evaluate the capacity of the resins to act as an affinity matrix, synthetic ligand for FKBP12 (SLF), which specifically and tightly binds to FKBP12, was used as a model system. The efficacy of the SLF-immobilized resins was evaluated by affinity purification of FKBP12 from both a cell lysate and a phage cDNA library. Among the resins tested in this study, SLFimmbolized PEGA resin was the most effective at capturing FKBP protein. This result suggested that the favorable swelling

2424 Bioconjugate Chem., Vol. 19, No. 12, 2008

Figure 8. Kinetic analyses of the binding between SLF and SLF-binding peptides by using the QCM apparatus. (A) Time course of the frequency change of the SLF-immobilized (red) or control (black) sensor chip, responding to the addition of FKBP12. Various concentrations of FKBP12 were added to the cuvette. The resulting overall concentrations of FKBP12 in the cuvette were as follows: 1, 7.2 nM; 2, 21.6 nM; 3, 50.3 nM; 4, 79.0 nM; 5, 107.7 nM; 6, 136.4 nM. (B) Time course of the frequency change of the SLF-immobilized (red) or control (black) sensor chip, responding to the addition of clone 2 peptide. Various concentrations of the peptide were added to the cuvette. The resulting overall concentrations of the peptide in the quvette were as follows: 1, 0.3 µM; 2, 0.8 µM; 3, 1.3 µM; 4, 1.8 µM; 5, 4.8 µM; 6, 6.8 µM.

properties of PEGA resin (16 mL/g) in aqueous media enabled affinity purification with high binding capacity and specificity. The swelling properties of the other resins are less favorable (e.g., 4 mL/g for TentaGel and NovaSyn TG resin in water). Furthermore, the hydrophilic nature of the PEGA matrix afforded easy access of the protein to the immobilized ligand on the resin. Our previous report showed that TentaGel, which is PEGcoated polystyrene beads, was an effective resin for purifying DNA polymerase β. DNA polymerase β was purified from a Molt4 nuclear extract by LCA-immobilized TentaGel. In the present study, TentaGel was found to be unsuitable for purifying FKBP12. Tamura and co-workers also failed to isolate FKBP12 from a cell lysate with FK506-immobilized TentaGel, although the background itself was very low (8). These results suggest that hydrophobic ligands are poorly displayed on the surface of TentaGel resin, probably because the ligands have a tendency to form hydrophobic interactions with the polystyrene core by distorting the long flexible PEG chains (Mw 2000-3000 Da) (8). The very long PEG chains of TentaGel are not suitable for a spacer as affinity matrix when the hydrophobic ligands are immobilized on TentaGel. By contrast, DNA polymerase β binds to LCA-conjugated TentaGel because the hydrophilic carboxylic acid groups of LCA might prevent direct interaction with the polystyrene core. Here, we demonstrate that SLF-immobilized PEGA resin can be used to purify FKBP12 from using Jurkat cell lysates with a low background. To our knowledge, this is the first case to exemplify that the target protein could be isolated from a cell

Kuramochi et al.

Figure 9. Langmuir isotherm plots for (A) FKBP12 and (B) clone 2 peptide binding with SLF-SAM (7) by QCM analyses.

lysate with a small ligand-immobilized PEGA resin. The hydrophilic PEG chains (Mw 800 Da) on the surface chains may act as a suitable linker not only to display the hydrophobic ligand on the surface but also to reduce nonspecific binding of proteins on the core. Auzanneau et al. reported that an antibody against Streptococcus Group A trisaccharide recognized the small molecule on PEGA resin (14). Sucholeiki et al. revealed that methotrexate-immobilized PEGA magnetic matrix specifically captured the folate binding protein from several kinds of proteins (bovine albumin, chicken albumin, folate binding protein, lysozyme, lactferrin, and lactoperoxidase precursor) (16). Taken together with our results, these observations indicated that PEGA resin could be applicable for affinity purification using small molecules with a variety of chemical properties as ligands. We also investigated the application of the resins for phage display screening. Recently, several groups have succeeded in determining the ligand-binding site of the target protein by this screening method (29-37), where an avidin-coated matrix with a biotinylated compound was generally utilized. We found that the T7 phage displaying FKBP12 specifically bound the SLF immobilized on PEGA resin and that the PEGA matrix as well as AffiGel showed better signal-to-noise ratio than Sepharose 4B. These observations indicate that the PEGA resin is a useful matrix not only for affinity purification of proteins but also for phase display screening. Using the SLF-immobilized PEGA resin, we successfully isolated 2 different clones out of the phage cDNA library derived from Colo205 cells. One clone (clone 1) encoded full length FKBP12 with the untranslated region, and the other phage clone (clone 2) displayed the 20 amino acid peptide (SLSCLWAELSGKLWPLPAAQ, clone 2 peptide). We verified that the dissociation constant of the clone 2 peptide was 5.8 µM using the QCM apparatus, whereas interaction of the peptide with the

PEGA Resin As an Affinity Matrix

acetylated disulfide was barely detectable (KD ) 10.1 mM). Although the interaction between the peptide and SLF was weaker than that between FKBP12 and SLF (0.027 µM), these results suggest that the peptide specifically binds to SLF. We also found that human SoxE proteins contain a region homologous to the clone 2 peptide. The SoxE proteins include Sox8, 9, and 10, which regulate transcription in various developmental processes, such as chondrogenesis, male sex determination, neural crest development, and gliogenesis (46-48). The 13 amino acids of SoxE proteins showing similarity to the clone 2 peptide are located in helix 2 of the HMG domain. This helix contacts the base pairs of the target DNA site and is followed by the C-terminal region of the HMG domain, including helix 3 and the C-terminal tail, which are required for interaction with numerous transcription factors (49-51). From these observations, we propose that SLF interferes with the binding between SoxE proteins and DNA and/ or that the protein-protein interactions with transcription factors are inhibited by the conformational change of the C-terminal region of SoxE proteins due to the binding of SLF to the helix 2 domain. Therefore, although SLF does not exhibit a significant cellular response, it is possible that SLF might block certain cell differentiation pathways in which the SoxE proteins are involved. In summary, we investigated the application of the resins used in solid-phase synthesis for affinity purification. Since resins used in solid-phase synthesis are chemically and physically stable, various approaches for synthesis and immobilization of small ligands are available by using the resins. Of the resins tested in this study, PEGA resin was the most effective for isolating FKBP12. This matrix enabled the isolation of FKBP12 from a cell lysate, and the identification of SLF-binding peptides from a phage cDNA library. Moreover, PEGA resin displayed as appropriate properties for affinity protein purification and phage display as AffiGel in our experimental conditions. Our study suggests that PEGA resin has great potential as a tool not only for the purification and identification of small-molecule binding proteins but also for the evolution and selection of peptides or antibodies that recognize target molecules. Further proteomics applications of PEGA resin are currently underway. Supporting Information Available: Binding competition assays of FKBP12 absorption on the SLF-immobilized PEGA resin (Figure S1), preparation of lithocolic acid-immobilized resins (Scheme S1), binding analysis between a mammalian DNA polymerase β and the lithocolic acid-immobilized resins by the Bradford method (Figure S2), preparation of glutathione (GSH)-immobilized resins (Scheme S2), binding analysis between glutathione S-transferase (GST) and the glutathione (GSH)-immobilized resins by the Bradford method (Figure S3), and alignment between the clone 2 peptide and human Sox 8-10 proteins (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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