Analytical Biochemistry 322 (2003) 164–169

Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin
Ling Ren,a Edith Chang,a Khadijah Makky,b Arthur L. Haas,b Barbara Kaboord,a and M. Walid Qoronfleha,*

Bioresearch Division, Perbio Science, 2202 N. Bartlett Avenue, Milwaukee, WI 53202-1009, USA b Biochemistry Department, Medical College of Wisconsin, Milwaukee, WI 53226, USA Received 6 May 2003

Abstract We have developed an affinity-precipitation technique to facilitate conducting glutathione S-transferase (GST) pull-down assays. The dehydrated immobilized glutathione resin format, when combined with microcentrifuge spin columns, is a powerful tool that enables the simultaneous performance of resin hydration, the binding of the GST fusion protein, and the pull-down step with the appropriate protein partner in a semihigh-throughput fashion (multiple samples processed at the same time). The entire assay process is shortened and recovery is enhanced when coupled with a spin-column format, providing a convenient way to study protein–protein interactions. We successfully tested the resin format/technique in three common pull-down applications utilizing radiolabeled, overexpressed, and activated endogenous interacting protein partners. Ó 2003 Elsevier Inc. All rights reserved.
Keywords: GST pull-down; Affinity-precipitation; Protein–protein interaction

The study of protein–protein interactions is a major topic in proteomics. Pull-down or affinity-precipitation assays using glutathione S-transferase (GST)1 fusion proteins have become a common approach to study protein–protein interactions [1]. GST pull-down assays have been used to identify new interacting protein partners for a known protein or to confirm the interaction within a protein pair that has been previously discovered by other methods such as yeast two-hybrid screening or phage display [2–4]. In a GST pull-down assay, the ‘‘bait’’ protein is expressed as a GST fusion protein and then purified and immobilized onto glutathione-coupled resin. Once bound, the bait is incubated with either a cell lysate or
* Corresponding author. Fax: 1-414-227-3759. E-mail address: (M. Walid Qoronfleh). 1 Abbreviations used: GST; glutathione S-transferase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Trisbuffered saline. RBD, Ras or Rho binding domain; BSA, bovine serum albumin; DTT, dithiothreitol; NP-40, Nonidet-P40; FBS, fetal bovin serum.

another protein mixture to ‘‘pull down’’ the ‘‘prey’’ protein(s). However, preparation and equilibration of the resin prior to each pull-down assay is time consuming. Furthermore, consistently dispensing small amounts of resin slurry is always a challenge. A dehydrated format of glutathione resin called SwellGel2 immobilized glutathione disk [5] has been developed to eliminate the equilibration step, to ensure the equal dispensing of resin for each GST pull-down assay, and to enable the simultaneous performance of multiple assays. Another advantage of SwellGel is that this format facilitates long-term storage without compromising its function [5]. Since the entire pull-down assay is conducted in a single spin column, buffer is efficiently separated from the resin, thus preventing resin loss during the washes. As a result, the entire assay process is shortened and recovery is enhanced [6]. In this study, several GST pull-down assays using different interacting partners were performed to show that SwellGel disks retain affinity activity, i.e., provide

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Briefly. Reagents used for resin activation and ligand attachment were of the highest chemical grade possible and were obtained from Pierce and/or Sigma. The PCR product was then cloned into pFLAG-CMV-2. Elutions were combined for each reaction and the protein concentra- . GST pull-down assays Pull-down of 35 S-labeled HsMDM2. at the EcoRI and BamHI sites. these types of disks are available commercially from Pierce Biotechnology. Samples were washed three times with 400 ll Wash buffer from the B-PER GST Spin Purification Kit (Pierce) and then eluted four times with 50 ll of 24 mM glutathione made in the same buffer. The PCR product was cloned into pDONR-1 vector and then subcloned into pDEST15 (GST fusion vector) utilizing Gateway Technology (Invitrogen). an ubiquitin-conjugating enzyme. Protein interactions were verified in three systems in which the prey protein source was either recombinant. tion was measured by Coomassie Plus Protein Assay Reagent Kit (Pierce). or transcribed/translated in vitro. and GST-Raf1-RBD The expression of GST fusion proteins in bacteria was induced with 1 mM isopropyl-b-D -thiogalactoside for 3 h. was transcribed and translated in vitro from pGEMHsMDM2K446R [9] using the TN T SP6 Quick Coupled Transcription/Translation System (Promega) in the presence of 1000 mCi/mmol [35 S]methionine (Amersham Biosciences). 15 nmol) was incubated with one immobilized glutathione SwellGel disk or 100 ll of immobilized glutathione resin (50% slurry) for 30 min at room Materials and methods SwellGel disk preparation Preparation of SwellGel disks has been described previously [5. The human aCP1 was amplified by PCR from aCP1 EST clone (ID No. temperature. Binding capacity determination The binding capacity of an affinity resin is largely dependent on experimental conditions. The SwellGel format when combined with microcentrifuge spin columns is a powerful tool to facilitate pull-down assays. Purified GST-Hsp53N (50 lg. Molecular cloning The N-terminal domain (amino acids 1–82) of human p53 (Hsp53N).14]. Both clones Hsp53N and Raf1-RBD were confirmed to be identical to the published sequences. Incyte Genomics). GST fusion proteins were purified and eluted using the BPER GST Spin Purification Kit (Pierce) according to the manufacturerÕs instructions. The bacteria expressing GST-Hsp53N or GSTE2EPF were lysed with B-PER Bacterial Protein Extraction Reagent II (Pierce). an oncoprotein. endogenous. Inc. 25 mM Tris and 150 mM NaCl.7]. Currently. / Analytical Biochemistry 322 (2003) 164–169 165 the same functional equivalence as traditional wet resin (dehydrated resin vs wet resin) yet with better assay performance results (reduced time and enhanced recovery).2) at a concentration of 5 mg/ml was used to set up samples with various protein amounts to test binding. The bacteria expressing GST-Raf1-RBD were lysed by sonication in TBS. and airflow) in a Cincinnati Sub-Zero chamber. pH 7. a mammalian transient expression vector (Sigma). a tumor suppressor. The dispensed resin was dried under tightly controlled conditions (time. was cloned as previously described [13. The pDEST15-Hsp53N or pGEX-4 T2-Raf1-RBD was transformed into Escherichia coli BL21 (DE3) (Novagen).L. humidity. 770771. GST-E2EPF . A stock solution of rGST in Tris-buffered saline (TBS. Ren et al. The purified GST fusion proteins were quantified using the Coomassie Plus Protein Assay Reagent Kit (Pierce). Therefore. 15 nmol) or GST (39 lg. A SwellGel disk was placed in each spin column and allowed to incubate at room temperature for 30 min with gentle rocking. Pull-down experiments used one disk of immobilized glutathione SwellGel (50 ll settled resin) per sample. These systems provide prey proteins in differing quantities and levels of complexity to validate the applicability of SwellGel disks. The 35 S-labeled human MDM2 (HsMDM2). The GST-E2EPF . The formulated slurry was dispensed into 384-well polypropylene plates using a multichannel pipette (Matrix) or a robotic liquid dispensing machine (a Zymark SciClone ALH500 instrument) to obtain 50 ll settled resin per well. Expression and purification of GST-Hsp53N. was amplified from human p53 cDNA [9] by polymerase chain reaction (PCR). The Ras binding domain (RBD) of human Raf1 [10–12] was amplified by RT-PCR from HeLa cells total RNA and cloned into pGEX-4 T-2 vector (Amersham Biosciences) at the EcoRI and XhoI sites. The resin was equilibrated in the appropriate buffer and necessary additives were added into the slurry [5]. chromatographic resins were obtained from Amersham Biosciences and then activated and coupled with reduced glutathione ligands [8]. both clarified bacterial lysate and purified. The GST-RhotekinRho binding domain (GST-Rhotekin-RBD) and the EZ-Detect Rho Activation Kit for pull-down assay were obtained from Pierce. recombinant GST (rGST) were utilized to examine the overall binding capacity of dehydrated resin and compare it to wet resin.

A disk equivalent to 50 ll of resin slurry weighs 6.5 mg of rGST from crude lysate (nonsaturating conditions) [5]. The resin was then washed four times with 400 ll buffer B.5% BSA. i. 0. eliminating the wash steps required when using wet resin. GSTE2EPF or GST was incubated with one immobilized glutathione SwellGel disk for 30 min at room temperature. 0. The proteins were eluted with SDS sample buffer. Consequently.2 Æ 0.5% NP-40. or glutathione resin (control) in the presence of buffer A (TBS containing 10% glycerol. The resin was washed four times with 400 ll buffer B (TBS containing 0. This is comparable to the stated binding capabilities of wet resins available from different vendors.166 L. After washing to remove unbound protein. Prior to transfection. Binding capacity determination of SwellGel disk (50 ll settled resin) using purified. and 5% glycerol) per dish. Results and discussion SwellGel disks can be produced in a variety of sizes to accommodate variable bed volumes and capacities. followed by treatment with 10% FBS at indicated time intervals. cells were serum starved for additional 24 h. GST-bound resin.5% NP-40 and 1 mM DTT). 1. dried disks. Half of the sample volumes (25 ll) were separated by 4–20% SDS–PAGE transferred onto a nitrocellulose membrane. . 1 mM DTT. 10 lg/ml) and the protein bands corresponding to Flag-aCP1 were detected using SuperSignal West Pico chemiluminescent substrate (Pierce). Ren et al. recombinant GST. At 24 h after plating. preparation/equilibration of slurry. / Analytical Biochemistry 322 (2003) 164–169 temperature.6 mg. if the volume of glutathione resin changes. the amount of GST bound to the resin will change. Proteins were eluted by adding 50 ll of 2Â SDS sample buffer and heating at 95 °C for 5 min. 1). separated on a 4–20% SDS–PAGE (Invitrogen) and transferred onto a nitrocellulose membrane.. The capacity and binding efficiency of SwellGel glutathione disks were compared to those of wet resin. The membrane was probed with anti-Flag M5 monoclonal antibody (Sigma. The clarified cell lysate (300 ll) was incubated with GST-Raf1-RBD (80 lg) and one immobilized glutathione SwellGel disk at 4 °C for 1 h. Therefore. streptomycin. GST-E2EPF -bound resin. Each 50 ll of resin volume made into a SwellGel disk has a maximum binding capacity of 1 mg of pure rGST (saturating conditions) (Fig. Pull-down of active Ras from NIH3T3 cells in response to serum. followed by fluorography.e. 2. NIH 3T3 cells were grown in 100-mm culture dishes in DulbeccoÕs modified EagleÕs medium (HyClone) that was supplemented with 10% FBS (HyClone). The uniformity of each SwellGel disk is determined by weighing a representative population of dispensed. GST-bound resin.7]. or the glutathione resin alone was incubated with 300 ll cell lysate (3 Â 106 cells) in the presence of 300 ll buffer A for 4 h at 4 °C. A549 cells were harvested at 24 h after transfection and lysed with M-PER Mammalian Protein Extraction Reagent (Pierce) according to manufacturerÕs instructions. The drying process removes excess water from the gel slurry. Cells were lysed in 500 ll buffer (TBS containing 5 mM MgCl2 . Since the SwellGel disks do not contain preservatives they can be rehydrated directly in the binding buffer or with the protein sample. The GST pull-down system is depicted in Fig. 1 mM DTT and Complete protease inhibitors (Roche)). pull-down results could be affected by variation in resin volume (see below). leaving a solid pellet containing hydrated chromatography beads held together by surface tension and hydrogen bonding [5. a 200 ll resin SwellGel disk is capable of purifying up to 1. Goat anti-mouse antibody conjugated with horseradish peroxidase (Pierce) was used as the secondary antibody and detection was performed with SuperSignal West Pico chemiluminescent substrate (Pierce). Note that one never binds everything loaded since there is always an equilibrium between bound and unbound GST ($80%) no matter how much excess glutathione is on the resin (Fig. and blotted with anti-Pan-Ras mouse monoclonal antibody. reticulocyte lysate containing 35 S-labeled HsMDM2 was added to GST-Hsp53N-bound resin. All of the GST pull-down experiments were performed in microcentrifuge spin cups using one disk of immobilized glutathione SwellGel (50 ll settled resin) per sample. Human lung carcinoma A549 cells were maintained in EagleÕs minimum essential medium supplemented with 10% fetal bovine serum (FBS) (HyClone). Pull-down of overexpressed Flag-tagged protein. The rehydration of the SwellGel disk and the binding of 1200 µg of eluted rGST 1000 800 600 400 200 0 0 200 400 600 800 1000 1200 1400 µg of loaded rGST Fig. Proteins were eluted in 50 ll SDS sample buffer and separated by 12% SDS–PAGE (Invitrogen). The binding reaction was performed at 4 °C for 4 h with gentle agitation. 1% NP-40. cells were plated and grown for 24 h to 90% confluency and transfected with pFLAG-aCP1 (see above) using Lipofectamine 2000 (Invitrogen). 1). 2 mM glutamine. and penicillin (100 U/ml).

These methods include the following: (1) in vitro transcription/translation (radiolabeled protein). The cDNA corresponding to aCP1 was cloned into a mammalian expression vector containing a Flag epitope tag and transiently expressed as a Flag-tagged recombinant protein in human lung carcinoma A549 cells..L. Using a GST fusion protein to pull down an in vitro transcribed/translated and 35 S-labeled interaction partner is a common approach in studying protein–protein interactions. The MDM2 oncoprotein is a ring finger ubiquitin ligase that plays a central role in the regulation of the p53 tumor suppressor protein. SwellGel disks were evaluated in three common pulldown applications. Fig.14. 2.7]. GST pull-down of HsMDM2 to compare the performance of wet resin vs SwellGel. 3. and promotes its degradation in many tumor cells [15]. GST-Hsp53N fusion protein (50 lg) and GST (39 lg) (equimolar amounts of protein. A yeast two-hybrid screen showed that the 40-kDa RNA binding protein aCP1 was a binding partner for the ubiquitin-conjugation enzyme E2EPF (unpublished results of K. MDM2 binds to p53. Makky and A. preventing the loss of resin between washes. Generally. blocks its activity as a tumor suppressor. 15 nmol each) were immobilized onto SwellGel Immobilized Glutathione or wet resin.19]. A signal above the basal level noted is considered specific to p53:MDM2 interaction [16]. Haas). 35 S-labeled human MDM2. GST-p53N but not GST specifically interacted with MDM2 although minor nonspecific interaction was observed. The entire assay can be processed in one microcentrifuge spin cup. i. Purified GST-p53N was prebound to a SwellGel Immobilized Glutathione disk or wet resin. Ren et al. / Analytical Biochemistry 322 (2003) 164–169 167 lysate containing in vitro expressed and 35 S-labeled HsMDM2. As shown in Fig. Pull-down using overexpressed protein Next. and (3) an activated endogenous form (native protein).e. Immobilized proteins were then incubated with rabbit reticulocyte lysate that contains in vitro transcripted/translated. This N-terminal domain of p53 was cloned and expressed as a GST fusion protein. The MDM2 binding domain of p53 has been mapped to the first N-terminal 82 amino acids [16]. . Assembly of spin columns used in GST pull-down assays. To confirm this interaction. GST fusion proteins to the SwellGel can be carried out simultaneously. The nonspecific interactions could be eliminated by higher-stringency washes (increasing salt concentration or including detergents). Pull-down using in vitro transcribed/translated protein The first model system examined was the interacting pair of human p53 and MDM2. The eluted pull-down protein complexes were separated by 12% SDS–PAGE and detected by fluorography. a multifunctional protein with a primary role in mRNA stability and translational silencing. The nature of nonspecific proteins binding to affinity resins is largely through charge and other noncovalent interactions which is very common [5. The findings of our experiments are discussed below.17] and RNA binding protein aCP1 [18. we examined the performance of SwellGel disks by analyzing the interaction of ubiquitin conjugation enzyme E2EPF [13. and then incubated with reticulocyte Fig. GST-E2EPF was used as a bait protein and prebound to SwellGel Immobilized Glutathione resin. 3. The GST fusion proteins are either immobilized on the resin prior to pull-down or incubated with the resin and the interaction partners concurrently. (2) overexpression in mammalian cells (recombinant protein). the pull-down strategy employs one of several protocols to obtain the prey protein source. the pull-down signal of 35 S-labeled MDM2 by GST-p53N using the SwellGel disk was the same as that using the wet resin (functional performance).

some nonspecific binding is observed and signal above basal level is considered specific (see comments above). 500 or 700 lg) was used in each pull-down assay. This biphasic response is a novel finding. 5. GDP-bound state and an active. 4. or the SwellGel disk alone (lane 5). Ras cycles between an inactive.21]. migration. / Analytical Biochemistry 322 (2003) 164–169 A Flag-tag-specific antibody was used to distinguish the tagged protein from the endogenous protein. Similar results were attained with wet resin (data not shown). Half of the eluted volume from each sample was analyzed by Western blotting using anti-Pan-Ras antibody. 350. Ren et al. Cell lysates (300 lg) were prepared at periodic times and incubated with GST-Raf1-RBD and a SwellGel Immobilized Glutathione disk. the Raf1-RBD can be used as a probe to specifically isolate active Ras. The families of Ras and Rho are of special interest as they influence the cellÕs response to the changing environment [22]. Half of the eluted pull-down product was analyzed by Western blotting using anti-Rho antibody. The lysate (300 lg) was incubated with GST-Raf1-RBD and one immobilized glutathione SwellGel disk. when immobilized on a resin. nontransfected lysate. The Flag-aCP1 was detected with anti-Flag tag M5 antibody. serum-starved NIH3T3 cells were stimulated by 10% serum over a 1-h period. Ras GTPase interacts with downstream effectors such as Raf1 [10.3]. These proteins have been shown to regulate proliferation. Fig. especially with low-affinity binding partners. So. The pulled down proteins were eluted and separated by 4–20% SDS–PAGE and then transferred onto a nitrocellulose membrane. Therefore.12]. Again. followed by treatment with 10% FBS at the indicated time. actin cytoskeleton reorganization. In our experiment. including binding of the GST fusion protein (bait) to the GSH resin and binding of the bait to the Fig. Like other G proteins. NIH 3T3 cell lysate (500 ll for each assay) was treated with GTPcS according to manufacturerÕs instruction for the EZ-Detect Rho Activation Kit (Pierce). GST-Rhotekin-RBD (250. apoptosis. the Flag-tagged aCP1 was overexpressed in A549 cells (lane 1) and interacted specifically with GSTE2EPF (lane 3) but not GST or glutathione resin alone (lanes 4 and 5. It is difficult to discern the signals where wet resin is used due to inconsistent resin dispensing coupled with resin loss during frequent washes (data not shown). GST pull-down of active Ras. GST pull down of active Rho. Upon binding to GTP. was used to pull down the active or GTP-bound Ras. GTP-bound state. Flag-aCP1 transfected cell lysate. The quantity of Rho protein pulled-down is influenced by the amount of GST fusion protein bound to the resin. This type of pull down assay is also very useful for mapping the interaction domains on the target protein in vivo [20. 4. GTP-Ras [23]. consistent. GST pull-down assays are widely used to confirm the results of protein–protein interactions obtained from other experiments. While small inconsistencies in resin volume may not be discernible larger ones could influence pull-down efficiencies. GST captured on SwellGel disk (lane 4). such as the yeast two-hybrid screen and phage display [2. Pull-down using activated endogenous protein Small GTPases serve as molecular switches in regulating a wide range of essential biochemical pathways in eukaryotic cells. biphasic activation of Ras was detected at 1 min and at 30–60 min in response to serum. As shown in Fig. transformation. Lane 1. i. the Raf1-RBD was expressed as GST fusion protein which. A549 cells were transfected with pFLAG-aCP1 plasmid.e.. . differentiation. Multiple GST pull down assays were performed to detect the activation of Ras in response to serum stimulation. uniform resin quantity is important.168 L. GST pull-down of Flag-tagged aCP1. 6. respectively). Half of the eluted pull-down samples (25 ll) and 20 lg of the cell lysate were analyzed by Western blotting using anti-Pan-Ras mouse monoclonal antibody. In the assay. Fig. and cell cycle progression [22]. NIH3T3 cells were serum-starved for 24 h. As shown in Fig. Cell lysates were then incubated with GST-E2EPF captured on immobilized glutathione SwellGel disk (lane 3). Every binding event is in equilibrium. 5. lane 2.

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