ABSTRACT
G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.
In recent years, functional proteins and random peptide libraries have been assembled on the surface of bacteria (14, 16, 21), bacteriophages (4, 7), viruses (15), and yeasts (30), allowing the manipulation of diverse molecules, which provides tremendous applications in environmental and biomedical fields, such as the identification of antigens for vaccine treatments (8) and reduction of toxic metals (14, 33). These techniques have been achieved by using anchor molecules for foreign proteins to be assembled on the surface of microorganisms.
Transmembrane proteins such as OmpA (21), LamB (14), and surface proteins on flagella (34) and spores (13) have been used as anchor molecules by fusion or insertion of foreign proteins into the exposed loops (8). A chimeric protein consisting of the first nine N-terminal amino acids of Escherichia coli lipoprotein (Lpp) and five transmembrane segments of OmpA was used as an anchor molecule (5, 9). Efficient assembly of large foreign proteins was accomplished by fusion into the C-terminal portion of Lpp-OmpA without loss of activity. These complexes become highly dependent on the structural properties of the inserted foreign protein domain, since the protein will be more constrained when inserted into a permissive site of an anchor molecule. Therefore, the selections of an anchor molecule and fusion site are very important for efficient protein assembly on microorganisms.
Magnetic bacteria synthesize intracellular particles of magnetite (Fe3O4), known as magnetosomes, which are aligned in chains and individually covered with a stable bilayer membrane, which mainly consists of lipid and protein (1, 10, 11, 20, 26). These structures, called bacterial magnetic particles (BMPs), have been utilized for a number of clinical applications as the supports of biomolecules. Protein assembly onto BMPs can be realized by fusion techniques involving anchor proteins isolated in Magnetospirillum magneticum AMB-1. The MagA protein (46.8 kDa) was isolated following transposon mutagenesis and identified as an integral iron transport protein that transports iron across the BMP membrane for magnetite synthesis (23). This transmembrane protein was previously used as an anchor molecule for assembling soluble proteins such as luciferase (24), acetate kinase (19), and protein A (18) onto BMPs. These nanosized biomaterials were developed for use in chemiluminescence immunoassays employing an automated robot system (32). However, MagA maintains a large hydrophobic domain, making it unsuitable for assembling membrane proteins such as G protein-coupled receptors (GPCRs). A 16-kDa protein, Mms16, which was the most abundantly expressed of the BMP membrane-specific proteins of M. magneticum AMB-1, was recently identified (26). This protein showed similar characteristics to a small GTPase involved in the formation of intracellular vesicles and is tightly bound to or anchored in the BMP membrane (25).
In this study, Mms16 was employed as a new anchor molecule on the BMP membrane. An assembly system of seven transmembrane domain proteins, GPCRs, on BMP surfaces has been developed by using Mms16. D1 dopamine receptor (D1R) was used as a model GPCR, and ligand binding using recombinant BMPs was investigated.
MATERIALS AND METHODS
Bacterial strains and culture conditions. Escherichia coli strain DH5α was used as a host for gene cloning. Cells were cultured in Luria-Bertani medium at 37°C containing ampicillin (50 μg/ml) or tetracycline (12.5 μg/ml). M. magneticum AMB-1 was microaerobically cultured in MSGM at 25°C as previously described (17). Microaerobic conditions were established by sparging the cultures with argon gas. Batch cultures were prepared in 2 volumes in 4-liter flasks. AMB-1 transformants were cultured under the same conditions containing ampicillin or tetracycline (5 μg/ml).
Construction of expression vectors.The plasmids pUM16 and pUMP16 were respectively derived from pUMG (6.4 kbp, Apr) (27). Plasmid pUM16 contains an NsiI site downstream of the sequence encoding mms16 promoter (pmms16) and mms16 (950 bp). The fragment was generated by PCR amplification with m16F (5′-GCTTTGCCAGTCGCTGCTGGAAGCGGTGG-3′) and m16R (5′-ATGCATCTTCTTGCCGGCCTTGGTGAAGACC-3′), using AMB-1 genomic DNA as a template. The PCR fragment was cloned into pGEMTeasy (Promega, Madison, Wis.), this plasmid was digested with EcoRI, and the fragment containing pmms16 and mms16 was cloned into EcoRI-digested pUMG. Plasmid pUMP16 containing a SpeI site downstream of the sequence encoding pmms16 was constructed as well as pUM16 by PCR amplification with m16F and pm16R (5′-ACTAGTCATGTTATTCCTCCAACC-3′). Plasmid pUM16 (7.5 kbp; Apr) was modified by replacing its Apr cassette with a Tetr cassette in pACYC184 (4.2 kbp, Tetr Cmr; Nippon Gene Co., Ltd., Tokyo, Japan). Both plasmids were digested with ScaI and XbaI and ligated. Plasmid pUM16t (8.9 kbp, Tetr) containing a cloning site (NsiI) was constructed.
For construction of plasmids pUMHA16, pUM16HA, pUML, and pUMML, ligated DNA fragments were prepared by PCR amplification with following primer pairs. The fragment encoding N-terminal hemagglutinin (HA)-tagged Mms16 was amplified with HA16F (5′-ACTAGTTATCCCTATGATGTCCCCGATTATGCCATGGCCGCCAAGCAGAG-3′) and HA16R (5′-ATGCATGCTTACTTCTTGCCGGC-3′) from AMB-1 genomic DNA for pUMHA16, and pUM16HA was constructed by ligating the fragment encoding the C-terminal HA-tagged Mms16 amplified with 16HAF (5′-ACTAGTATGGCCGCCAAGCAG-3′) and 16HAR (5′-TTAGGCATAATCGGGGACATCATAGGGATACTTGCCGGC-3′). The gene encoding luciferase was amplified with LF (5′-ACTAGTATGGAAGACGCC-3′) and LR (5′-TTACAATTTGGACTTTCCGC-3′) from pGV-SC (Toyo Ink Co. Ltd., Tokyo, Japan) for pUML. Finally, the gene encoding the fusion protein MagA-luciferase was amplified with MLF (5′-ACTAGTATGGAACTGCATCATCCCG-3′) and LR from pKML (24) for pUMML. Each fragment was cloned into pGEMTeasy. After digestion of these plasmids with SpeI (underlined), the fragments were inserted into SpeI-digested pUMP16.
For construction of pUM16L, the luciferase gene was amplified with 16LF (5′-ATGCATATGGAAGACGCCAAAAACATAAAGAAAGG-3′) and 16LR (5′-ATGCATTTACAATTTGGACTTTCCGCCCTTCTTGGC-3′) from pGV-SC. For pUM16D1 (10.2 kbp, Tetr), the intronless gene encoding human D1R (1,356 bp) was amplified with D1RF (5′-ATGCATATGAGGACTCTGAACACCTC-3′) and D1RR (5′-ATGCATTTATCAGGTTGGGTGCTGAC-3′) from the human genomic gene and then cloned into pGEMTeasy. Both plasmids were digested with NsiI (underlined), and the fragments encoding luciferase or D1R were cloned into pUM16 or pUM16t digested with NsiI.
The above plasmids and the previously constructed plasmid, pKML, were transferred into wild-type M. magneticum AMB-1 by electroporation (27).
Separation of BMPs and cell membrane from AMB-1.Eight liters of stationary-phase culture of M. magneticum AMB-1 was harvested by centrifugation (10,000 × g, 10 min, 4°C), resuspended in 20 ml of phosphate-buffered saline (PBS; pH 7.4), and disrupted by three passes through a French press at 1,500 kg/cm2 (Ohtake Works Co., Ltd., Tokyo, Japan). The BMP and disrupted cell fractions were separated magnetically with a columnar neodymium-boron magnet. The collected BMPs were washed with HEPES (10 mM) buffer several times and used for assays. The protein profile of BMP membrane proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The BMP membrane fraction was dissolved in 8 M buffered urea (containing 50 mM Tris, 10 mM dithiothreitol, pH 8.5). The membrane proteins were incubated with equal volume of 2× SDS-sample buffer, and the sample was run on an SDS-PAGE gel (12.5% polyacrylamide). The gel was stained with Coomassie brilliant blue R-250.
The cell membrane was fractionated as previously described (23). The disrupted cell fraction was centrifuged at 5,000 × g for 20 min to remove unbroken cells. The supernatant was ultracentrifuged at 96,600 × g for 1 h, and the obtained pellet was washed twice with HEPES buffer and resuspended in PBS buffer. The collected BMPs were washed with HEPES (10 mM) buffer several times and used for assays.
The concentration of BMPs in suspension was estimated by optical density at 660 nm, using a spectrophotometer (UV-2200; Shimadzu, Kyoto, Japan). A value of 1.0 corresponded to 172 μg of BMPs/ml (dry weight).
Expression of HA-tagged Mms16 on BMPs and its evaluation.Wild-type AMB-1 and transformants harboring pUM16HA and pUMHA16 were harvested and disrupted. The BMP fraction was washed with HEPES buffer 10 times. Recombinant BMPs (100 μg) were incubated with rhodamine-labeled anti-HA antibody (5 μg/ml; Beringer) for 60 min with pulsed sonication (15-s pulses at 10-min intervals), separated magnetically, and washed three times with PBST (PBS containing 0.05% Tween 20). Fluorescence intensity of BMPs (50 μg/100 μl) was measured with a fluorescence plate reader (FLUO Star Galaxy; BMG Labtechnologies, Ltd.).
Measurement of luciferase activities of recombinant BMPs and cell membrane.Wild-type AMB-1 and transformants harboring pUML, pUM16L, pUMML, and pKML were harvested and disrupted. The extracted BMPs were washed with HEPES until the luminescence intensity on the surface of BMPs was stable. The luminescence intensity of BMPs or cell membrane was measured with a Pika gene luminescence kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Resuspended BMPs (100-μl sample, 500 μg of BMPs/ml) and cell membrane proteins (100-μl sample, 10 μg of proteins/ml) were respectively mixed with 100 μl of luminescence substrate solution, and the luminescence intensity was measured with a Lucy-2 luminescence reader (Aloka, Co. Ltd., Tokyo, Japan).
Expression analysis of D1R on recombinant BMPs.Anti-human D1R antibody (anti-D1R) was used to evaluate the expression of D1R. A sample containing 100 μg of recombinant BMPs was washed and incubated with anti-D1R solution (1:500; Calbiochem-Novabiochem Corporation, Bad Soden, Germany) for 1 h at room temperature (RT) with pulsed sonication. The BMPs were then magnetically separated from the reaction mixture and washed three times with 200 μl of PBST. A 100-μl solution of alkaline phosphatase (ALP)-conjugated anti-rabbit immunoglobulin G antibody (1:2000; Sigma) was added as secondary antibody and incubated for 1 h at RT. After 3 washings, BMPs were mixed with 100 μl of Lumi-Phos 530, including lumigen PPD—4-methoxy-4-[3-(phosphonooxy)phenyl] spiro [1,2-dioxeteane-3,2′ adamantine], disodium salt (3.3 × 10−4 M)—as a luminescence substrate for ALP, and the luminescence intensity was measured with a luminescence reader. Wild-type BMPs were used as a control.
Ligand binding to recombinant BMPs.Ligand binding experiments with BMPs from AMB-1 transformants harboring pUM16D1 were performed using [N-methyl-3H]SCH23390 (Amrsham Pharmacia Biotech). Aliquots of 100 μg of BMPs from AMB-1 transformants or the wild type were added in 1.0 ml of binding buffer (50 mM Tris-HCl, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, 4 mM MgCl2, 120 mM NaCl) and incubated for 1 h at RT in the presence of 1 nM [3H]SCH23390. Separation of free ligand was achieved by rapid filtration through GF/B filters, followed by four washes with 5 ml of chilled 50 mM Tris-HCl, pH 7.4. Bound radioactivity was determined in a liquid scintillation counter.
For saturation binding experiments, the Millipore MultiScreen separations system was used according to the protocol described by the manufacturer. MultiScreen filtration plates (DV plate; Millipore) consisting of 96 wells were used for an assay involving reaction and separation of ligand binding. Recombinant BMPs (5 μg/well) or membrane proteins (0.02 μg) from Sf9 cells infected with baculovirus to express the human recombinant D1R (Research Biochemicals International, Natick, Mass.) were used as samples for this binding assay. The samples were incubated for 1 h at RT with the indicated concentrations of [3H]SCH23390 in a reaction volume of 200 μl. Nonspecific binding was determined in the presence of 1 μM SCH23390.
Fluorescence competitive binding assay.A competitive binding assay was performed using BODIPY FL SCH23390 (Molecular Probes, Inc., Eugene, Oreg.). A 50-μg aliquot of recombinant BMP was resuspended in 100 μl of BODIPY-labeled SCH23390 (10−7 M), and nonlabeled dopamine (Sigma) was added to set binding competition. The suspension was incubated for 1 h at RT and washed with 200 μl of Tris-HCl buffer to remove free ligands. The BMPs were washed five times and resuspended in 100 μl of binding buffer. Fluorescence intensity was measured by using a fluorescence microplate reader.
RESULTS
Expression of HA-tagged Mms16 on BMPs.In order to develop a new anchoring system for assembling functional proteins onto BMPs, an abundantly expressed small protein, Mms16, was selected among BMP membrane proteins by SDS-PAGE (Fig. 1A). Mms16 sequence and function were previously characterized (25, 26), but the mechanism by which it is anchored onto the BMP membrane is not clear. An appropriate fusion site N or C terminal to the anchor molecule Mms16 was examined by using HA tag. HA-tagged Mms16 on BMPs was detected with rhodamine-labeled anti-HA antibody. Recombinant BMPs, 16HA and HA16, extracted from transformants harboring plasmid pUM16HA (C-terminal HA tag) and pUMHA16 (N-terminal HA tag), showed higher fluorescence intensity than wild-type BMPs (Fig. 1B). This result indicates that heterologous Mms16 proteins were expressed on the BMP surfaces. Higher fluorescence intensity was obtained in 16HA than HA16. This indicates that the C-terminal fusion of Mms16 was more effective for assembling foreign proteins. The N-terminal part of Mms16 is probably important for anchoring, and the association of Mms16 with the membrane on BMPs was inhibited by the fusion. Based on these results, Mms16 was selected as an anchor molecule, and foreign proteins were fused at the C-terminal portion in subsequent experiments.
Evaluation of Mms16 as an anchor molecule for protein assembly onto BMP membrane. (A) SDS-PAGE of BMP membrane proteins. Forty micrograms of protein was applied to the gel. Standard molecular mass makers (in kilodaltons) were used. An arrowhead indicates Mms16. (B) Expression of HA-tagged Mms16 on BMPs. Fluorescent intensity (expressed in arbitrary units [a.u.]) was determined by immunoassay using rhodamine-conjugated anti-HA antibody when 50 μg of BMPs was used in 100 μl of PBST. WT, BMPs from wild-type AMB-1; 16HA, BMPs from AMB-1 transformant harboring pUM16HA; HA16, BMPs from AMB-1 transformant harboring pUMHA16.
Efficiency of anchoring of Mms16 and MagA onto BMP membrane.The efficiency of anchoring of Mms16 onto BMPs was compared with that of MagA. MagA, which has been used as an anchor molecule previously, is a transmembrane protein and has large hydrophobic domains, while Mms16 is a small hydrophilic protein. These different properties may affect the efficiency of anchoring onto BMPs. Luciferase, Mms16-luciferase, and MagA-luciferase were expressed in AMB-1 transformants under the control of the mms16 promoter. Localization of luciferase was verified by luminescence intensity. BMPs were extracted from the cellular debris and washed until a constant luminescence intensity was obtained before the luciferase activities on BMPs were determined. Recombinant BMPs extracted from the cells harboring pUML (no anchor molecule) did not show significant luminescence (Fig. 2A), while BMPs harboring pUM16L (Mms16 anchor), pUMML (MagA anchor), and pKML (MagA anchor) showed 10 times more luminescence, indicating that luciferase was localized on BMP membrane where the Mms16 or MagA was anchored. Furthermore, the luminescence intensity of BMPs extracted from cells harboring pUM16L was 1.8 times higher than that from cells harboring pUMML (Fig. 2A). The small hydrophilic protein Mms16 was anchored onto BMPs more efficiently than MagA under the control of the same promoter.
Efficiencies of anchoring Mms16 and MagA onto BMP membrane and cell membrane. Luminescent intensity of BMPs (50 μg) (A) and cell membrane (1 μg) (B) was determined with a luminescence reader (Lucy-2) after addition of luciferin solution (100 μl). (C) Schematic diagram of each plasmid. Solid, shaded, and white blocks indicate genes encoding luciferase, Mms16, and MagA, respectively. Shaded and white triangles indicate the promoters of mms16 and magA, respectively. The numbers 1 to 4 indicate the following: 1, pUML (promoter pmms16 and no anchor); 2, pUM16L (promoter pmms16 and anchor Mms16); 3, pUMML (promoter pmms16 and anchor MagA); 4, pKML (promoter pmagA and anchor MagA). Plasmids 1, 2, and 3 were derived from pUMG (27), and plasmid 4 was derived from pRK415 (24).
Focusing on the cell membrane, luminescence intensity was obtained from the cells harboring pUMML and pKML ligated with MagA (Fig. 2B). Luciferase was expressed with various intensities in different parts of the cell depending on the anchor molecules. Furthermore, the integration of MagA-luciferase into the cell membrane caused a slight growth inhibition in the AMB-1 transformant (data not shown).
Expression analysis of D1R on BMPs with Mms16 by antibody and antagonist binding.The expression of functional soluble proteins onto BMPs by using a hydrophobic protein, MagA, has been reported. In this study, the GPCR D1R was assembled on the BMP surface by using a small hydrophilic anchor protein, Mms16. The procedure for assembling D1R onto BMPs is shown in Fig. 3A. Plasmid pUM16D1 harboring a gene fusion of Mms16 and D1R was introduced into wild-type AMB-1. The recombinant BMPs were magnetically separated from the cell debris and washed several times. Localization of D1R on recombinant BMPs was assayed by immunodetection with anti-D1R, which recognizes the N-terminal side (ligand binding side) of D1R. ALP-conjugated anti-rabbit immunoglobulin G was used as a secondary antibody as shown in Fig. 3B, higher luminescence intensity was obtained on the surface of recombinant BMPs than in the wild type, indicating that Mms16-D1R fusion protein was assembled onto the BMP surface. Small hydrophilic proteins, like Mms16, are useful as an anchor molecule for the efficient anchoring of Mms16-D1R and the accessible location of the D1R ligand binding site on the BMP surface.
Expression analysis of D1R on BMPs by antibody and antagonist binding. (A) Schematic diagram for preparation of recombinant BMPs and assembly of D1R. In step a, a plasmid (pUM16D1) containing a gene fusion for Mms16 and D1R expression was transformed into wild-type AMB-1. In step b, the AMB-1 transformant harboring pUM16D1 was disrupted to release recombinant BMPs. In step c, recombinant BMPs were magnetically separated and purified by stringent washing. D1R was assembled on recombinant BMPs. (B) Expression analysis of D1R was performed with anti-D1R antibody by a method derived from enzyme-linked immunosorbent assay of BMPs extracted from AMB-1 (bar 1) and BMPs extracted from an AMB-1 transformant harboring pUM16D1 (bar 2). Luminescence intensity was determined with the secondary ALP-conjugated antibody and Lumiphos 530. (C) Binding of [3H]SCH23390 to the surfaces of the BMPs shown in bars 1 and 2. A sample of 100 μg of BMPs was used for each assay. Results represent the average with standard deviations from more than three experiments.
The activity of D1R expressed on the recombinant BMPs was confirmed with the antagonist [3H]SCH23390. Specific binding of the antagonist to the recombinant BMPs is shown in Fig. 3C. The specific binding to the recombinant BMPs indicates that D1R was assembled in a lipid membrane environment on the BMP surface and possessed the ability to bind to its ligand. Nonspecific binding of [3H]SCH23390 to wild-type BMPs was also observed. This binding might be caused by the assay procedure, which used a large number of BMPs that aggregated on the filter.
Saturation binding analysis was performed to assess the affinity of antagonist [3H]SCH23390 to D1R assembled on BMPs (D1R-BMP complexes) (Fig. 4A). D1R-BMP complexes displayed a single affinity for the [3H]SCH23390 with a Kd value of 9.7 nM. This is in good agreement with the value obtained from D1R prepared from cell membranes of Sf9 cells by using the baculovirus expression vector system (Kd = 2.4 nM). The D1R expressed in the prokaryotic M. magneticum AMB-1 cells had similar affinity to D1R expressed in the eukaryotic cells. This indicates that other human GPCRs can be expressed on BMP surface as well.
Specific saturation binding of a radiolabeled D1R-specific antagonist [3H]SCH23390 to recombinant BMPs extracted from AMB-1 transformants harboring pUM16D1.
Competitive binding assay of dopamine using fluorescent antagonist BODIPY-labeled SCH23390.For the development of a fully automated ligand screening system using D1R-BMP complexes, a competitive binding assay was performed by using a fluorescent antagonist with magnetic separation. In the presence of BODIPY-labeled SCH23390, various concentrations of the agonist dopamine were added, and a competitive binding assay was performed on recombinant BMPs. Fluorescence of magnetically separated BMPs was measured after several washings. As shown in Fig. 5, the increase of competing chemical concentrations decreased the fluorescence of BMPs. This competitive binding assay can also be adapted for use with other ligands of D1R.
Fluorescence competition binding analysis of dopamine to D1R on BMP surfaces. Recombinant BMPs were incubated with 10−7 M BODIPY-labeled SCH23390. Concentrations of dopamine, a D1R agonist, are indicated.
DISCUSSION
In this study, a novel system for protein assembly onto nanobiomagnetic particles has been developed, using a recently identified BMP membrane protein, Mms16, as an anchor molecule (26). MagA was originally isolated following transposon mutagenesis and was used as an anchor molecule in previous studies. However, MagA had very low expression and was difficult to observe in SDS-PAGE gels. On the other hand, Mms16 is a small protein abundantly expressed in the BMP membrane of M. magneticum AMB-1 (Fig. 1A). Mms16 was therefore selected as a candidate for anchor molecules and analyzed in this study.
From luciferase fusion studies, higher luminescence intensity was obtained from BMPs with Mms16 as an anchor molecule of luciferase than with MagA (Fig. 2A). One of the reasons for this result may be the total amount of expressed fusion proteins that depend on the molecular size and character of expressed proteins. Even though the same vector construct and promoter were employed, expression properties were sometimes different. Similar results were reported when LamB or OmpA was fused to different sizes of foreign peptide and expressed on E. coli cell surfaces. Increasing the peptide size decreased the expression of fusion proteins (8). A smaller fused protein like Mms16 (16 kDa) is more likely to be expressed than a larger protein like MagA (46.8 kDa). Furthermore, MagA fusions were expressed not only on the BMPs but also in the cytoplasmic membrane fraction (Fig. 2B). High heterologous expression on the cytoplasmic membrane occasionally leads to growth inhibition (3). Differences in the growth rates between the transformants harboring pUM16L expressing Mms16-luciferase and pUMML expressing MagA-luciferase were caused by the biochemical properties of the anchor molecules. Thus, a small-size protein like Mms16 as an anchor molecule is effective for functional protein assembly on BMPs and offers a versatile detection system.
The constructed BMP-Mms16 assembly system is applicable to several functional foreign proteins like single-chain Fv, random peptide libraries, metal binding peptide, and receptors. This study presents a novel system for GPCR assembly onto BMPs. GPCR represents one of the most predominant families of transmembrane proteins and is a prime target for drug discovery. Various challenges to ligand screening of GPCRs have been undertaken; however, these proteins are generally expressed at very low levels in the cell and are extremely hydrophobic, rendering the analysis of ligand interaction very difficult. GPCRs have been prepared through reconstitution with lipids or detergents on liposomes (6, 12), on chip surfaces for surface plasmon resonance detection (28, 29), on silica particles (31), and on paramagnetic beads (2, 22). Although lipids and detergent conditions can occasionally be found that allow the native structure to be maintained in solution, this is an empirical and frequently time-consuming process. Consequently, ligand binding analysis of GPCRs is commonly conducted with whole cells. In all cases, analysis of GPCRs requires a lipid or an environment that imitates lipids to maintain the native structure and function.
In this study, we have demonstrated an assembly system of D1R onto the surfaces of BMPs through genetic manipulations. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants harboring pUM16D1, and after several washings, the complexes were ready to use for analysis. This system efficiently obtains the native conformation of GPCRs without detergent solubilization, purification, and reconstitution after cell disruption. Moreover, BMPs are good biomaterials for a fully automated ligand screening system that uses magnetic separation. This facilitates rapid buffer exchange and stringent washings and reduces nonspecific binding, which interferes with analysis. This novel system provides advantages for studying various membrane proteins, which are usually difficult to assay.
ACKNOWLEDGMENTS
We are grateful to R. Calugay for helping out with the English in the manuscript.
This work was funded in part by a Grant-in-Aid for Specially Promoted Research 2, no. 13002005, from the Scientific Research for the Ministry of Education, Culture, Sports, Science and Technology of Japan and as part of the 21st Century Center of Excellence (COE) program of “Future Nano-Materials” research and an education project through Tokyo University of Agriculture & Technology. T. Yoshino thanks the Japan Society for the Promotion of Science (JSPS) for financial support.
FOOTNOTES
- Received 19 September 2003.
- Accepted 14 January 2004.
- Copyright © 2004 American Society for Microbiology
