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  • S j V W Ch J li Fl t h K i ti H il Wi l K dli ki K i L it Lif T h l i 5791 V All W C l b d CA 92008 USA

    Expression, Capture and Display of Native GPCRs on Soluble Mammalian Membrane Particles

    Figure 5. GPCRs in MembranePro™ Particles Display Pharmacologically Relevant Ligand Binding Activity withHigher Receptor Density Compared to Cell Membranes

    A. Beta-2 Adrenergic Serotonin 5HT1a

    MembranePro™Particles

    B S t i 5HT15060708090

    100110

    Total BoundS ifi B dB

    ound

    (pm

    ol/m

    g)

    5HT1a

    40506070

    nt

    Figure 4. Physical Characterization of MembranePro™ Particles

    A. B.

    Sanjay Vasu, Wen Chen, Julia Fletcher, Kristin Huwiler, Wieslaw Kudlicki, Kevin Lowitz • Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • USA

    ABSTRACT

    The study of mammalian membrane proteins is currentlyhindered by the lack of appropriate tools to produce andisolate membrane proteins in a condition and formatamenable to functional analysis. Crude cell membranescurrently serve as the gold standard material for in vitroh t i ti f b t i ti it H h

    ultracentrifugeto isolate crudemembranes

    MATERIALS AND METHODS

    Figure 1. Cell Membrane Preparation

    30

    40

    50

    Total Bound

    Bou

    nd (p

    mol

    /mg)

    B. Serotonin 5HT1a

    CellMembranes

    0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50

    1020304050 Specific Bound

    Non-Specific Bound

    BmaxKd

    Total Bound98.230.1871

    Specific Bound83.690.1406

    [3H]-WAY-100635 (nM)

    Rad

    ioac

    tivity

    B

    010203040

    114 126 139 154 170 188

    diameter (nm)

    coun

    Isolated particles were physically examined by electron microscopy (panel A) anddynamic light scattering (panel B). In panel A, particles were dried on a copper gridand negatively stained with uranyl acetate. Samples were imaged on a JEOLelectron microscope at 6000x magnification. Size bar is 200 nM. In panel B, particleswere diluted and analyzed in a Brookhaven90 particle analyzer by laser lightscattering. Calculated particle diameter (assuming a spheroid conformation)centered around 139 nm.

    characterization of membrane protein activity. Here we havedeveloped a mammalian functional protein expression (FPE)system, MembranePro™ FPE System to produce cellmembrane virus-like particles (VLPs) containing G protein-coupled receptors (GPCRs) captured from the cell plasmamembrane. We show that GPCRs which are concurrentlyexpressed in the cell are captured on the particles. Wedescribe conditions for membrane protein expression andcapture which optimize yield of GPCR activity. Methods forparticle harvesting are compared, and we demonstrate ahigh-efficiency method for rapidly isolating particles whichb passes the m ltiple centrif gation steps req ired for

    48 hours post transfection,decant culture media

    add MembranePro™Precipitation Mix

    low speed centrifugationto pellet particles

    10 - 4 10 - 3 10 - 2 10 - 1 10 0 10 1 10 2 10 3 10 4 10 5-20

    0

    20

    40

    60

    80

    100

    120

    5-CT

    5-HT

    Methiothepin

    Spiperone

    [Cold Ligand] (nM)

    Spec

    ific

    Bou

    nd (%

    )

    scrape cells to harvest and pellet homogenize pellet cell debris

    Figure 2. MembranePro™ Particle Isolation

    0 5 10 15 20 25 300

    10

    20 Specific BoundNon-Specific Bound

    BmaxKd

    Total Bound40.290.9315

    Specific Bound39.680.9345

    [3H]-DHA (nM)

    Rad

    ioac

    tivity

    B

    2

    4

    6

    8

    10

    Total BoundSpecific BoundNon-Specific Bound

    oact

    ivity

    Bou

    nd (p

    mol

    /mg)

    0.5

    1.0

    1.5

    2.0

    2.5

    3.0

    3.5

    Total BoundSpecific BoundNon-Specific Bound

    oact

    ivity

    Bou

    nd (p

    mol

    /mg)

    Panel A: In order to compare the saturation and competition binding kinetics of GPCRs from MembranePro™ particles and membranes, weisolated MembranePro™ particles or crude cell membranes from 293FT cells expressing beta-2 adrenergic and serotonin 5HT1a receptors.A fixed non-saturating level of protein was challenged with increasing concentrations of tritiated ligands in the presence or absence of coldcompetitor as indicated in the figure. Bound ligand was determined by collecting and washing samples by vacuum on filter plates followed byscintillation counting. In the case of each GPCR, Kd values were nearly identical between MembranePro™ particles and cell membranesindicating the two sources of GPCRs are pharmacological equivalent with respect to Kd. Bmax values, however, reflect the higher receptordensities captured on some of the particles (up to 30-fold higher than membranes, see red arrows).

    ce te ed a ou d 39bypasses the multiple centrifugation steps required formembrane fraction isolation. Negative stain electronmicroscopy and dynamic light scattering techniques revealthe particles have a uniform size distribution. Antagonistsaturation binding and competition assays reveal that themembrane particles offer higher receptor density than crudemembrane fractions while maintaining pharmacologicalequivalence with corresponding native cellular receptors.This technology provides a critical new tool supportingapplications in basic membrane protein research and can beused downstream for drug discovery, high-throughputscreening and antibody characterization

    deca t cu tu e ed aand remove cell debris

    Precipitation Mix to pellet particles

    C.Crude cell membranes and MembranePro™particles were analyzed by SDS-PAGE andstained with coomassie blue (panel C). As thecrude membrane sample containsintracellular membranes as well as totalplasma membrane, it contains a large numberof integral and membrane-associatedproteins. In contrast, as the particles aresecreted, they lack these contaminants. As aresult, in a particle sample that contains only

    Preparation of crude cell membranes is labor-intensive,involving the harvesting of cells, dounce homogenization,separation of cell debris from membranes and finally washesand differential centrifugation to crudely segregate plasmamembrane. The MembranePro™ protocol circumvents thelabor and manipulations of this process with a simplifiedworkflow involving precipitation of MembranePro™ particlesfrom culture media. Briefly, culture media is decanted 48hours post transfection (GOI/pEF6 TOPO® plasmid DNA,MembranePro™ Reagent and Lipofectamine™ 2000 into T-

    0 5 10 15 20 25 300

    BmaxKd

    Total Bound8.0760.8239

    Specific Bound7.1840.7083

    [3H]-DHA (nM)

    Rad

    io

    0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50.0

    BmaxKd

    Total Bound3.1680.1514

    Specific Bound2.8140.1306

    [3H]-WAY-100635 (nM)

    Rad

    io

    p p ( p g )

    Panel B: 5HT1a MembranePro™ particles were incubated with saturating levels of 3H-WAY-100635 followed by competition with coldagonists and antagonists over a wide concentration range. Cold compounds competed hot bound ligand in order of affinity indicating specificand pharmacologically relevant binding.

    Figure 6. Scalable Production of MembranePro™ Particles in FreeStyle™ 293-F Suspension Adapted Cells

    MembranePro™Particles fromFigure 3 MembranePro™ Precipitation Mix Performance

    Primary human fibroblasts

    screening and antibody characterization.

    INTRODUCTION

    Particle formation can be initiated in cells by expressing theviral core protein, gag. Gag protein cores (indicated in lightblue) bud from the cell under lipid rafts in the plasmamembrane, capturing and displaying raft contents asparticles are secreted into the culture medium. Concurrenttransient expression of membrane proteins (indicated in red)

    total protein (μg): 2.3 9spec. bound (cpm): 8987 6472spec. act. (cpm/μg): 3907 719

    *Cell membranes and particles were made from cellsexpressing muscarinic M1 receptor. Radioactiveligand = 3H-scopolamine.

    2.3μg of total protein, the only clearly visibleband is the gag core protein. Additionally, asparticles capture predominantly receptor-richlipid rafts, the particles exhibit significantlyhigher specific binding activity.

    MembranePro Reagent and Lipofectamine 2000 into T175 flask of 293FT cells) and clarified by low-speedcentrifugation. Clarified media is combined with 1/5th volumeMembranePro™ Precipitation Mix and incubated overnight at4oC. Particles are then recovered by low-speed centrifugationin a clinical centrifuge and resuspended in buffer of choice(e.g. PBS) for assay or storage.

    RESULTSSerotonin 5HT1a

    SourceBmax

    (pmol / mg)Protein / Reaction

    (μg / well)

    MembranePro™ Particles - 13.7 0.510

    15

    20

    Total BoundSpecific Boundy

    Bou

    nd (p

    mol

    /mg)

    FreeStyle™ 293-F

    The FreeStyle™ 293-F suspension adapted cell line demonstrates high transfection efficiency at large volumes combining 293fectin™transfection reagent and MembranePro™ Reagent facilitating easier large scale VLP production without media change. The resultingserotonin 5HT1a MembranePro™ particles from this alternative protocol resulted in a higher receptor density compared to cell membranes.

    Figure 3. MembranePro Precipitation Mix Performance

    A. B.

    Panel A: To determine the efficiency of particle isolation using MembranePro™ Precipitation Mix we compared particle yield by precipitation and ultracentrifugation

    transient expression of membrane proteins (indicated in red)which localize to the rafts (signaling proteins, GPCRs and ionchannels) allows capture of these recombinant proteins inessentially native context. As the expression vector (pEF6-TOPO®) contains no viral sequences or packaging signals,no viral RNA is packaged into MembranePro™ particles.

    Ultracentrifugation vs. MembranePro Precipitation Mix

    Ultracentrifuge MembranePro Positive Negative0

    2000

    4000

    6000

    - atropine+ atropine

    3 H-s

    copo

    lam

    ine

    (cpm

    )

    Yield vs. Particle Concentration

    1x 0.2x 0.1x 0.05x0

    50000

    100000

    150000

    200000

    Sample Dilution

    Part

    icle

    Yie

    ld (p

    g/m

    l)

    REFERENCES

    FreeStyle™ 293-F

    Cell Membranes 1.09 10

    0 5 10 15 20 250

    5

    pNon-Specific Bound

    [3H]-WAY-100635 (nM)

    Rad

    ioac

    tivity

    Panel A: To determine the efficiency of particle isolation using MembranePro Precipitation Mix, we compared particle yield by precipitation and ultracentrifugation.Duplicate preparations of muscarinic M1 receptor MembranePro™ particles were made and the media harvested. Half of each preparation was isolated usingMembranePro™ Precipitation Mix and the other half pelleted by ultracentrifugation (100,000xg for 2.5hrs). Isolated particles were quantified by ligand (3H-scopolamine) binding activity in the presence or absence of cold competitor (atropine). Precipitation provided equivalent yield as ultracentrifugation. Positive control =M1 membranes, neg control = untransfected cell membranes.

    Panel B: Yield of particles or precipitation efficiency is minimally impacted unless particle production deteriorates significantly. Duplicate MembranePro™ reactionswere split into 4 and diluted with media as indicated in panel B prior to precipitation. Particle yield from each sample was quantified by p24 assay (Zeptometrix).

    Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com

    Bouamr, F., Garnier, L., Rayne, F., Verma, A., Rebeyrotte, N., Cerutti, M., and Mamoun, R., (2000). Differential budding efficiencies of human T-cell leukemia virus type 1 (HTLV-1) gag and gag-pro polyproteinsfrom insect and mammalian cells. J. Virology 278, 597-609.

    Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S. (1999) Lipofectamine™ 2000 Reagent for Rapid, Efficient Transfection of Eukaryotic Cells. Focus 21, 54-55

    Garnier, L., Ravallec, Ml., Blanchard, P., Chaabihi, H., Bossy, J-P., Devauchelle, G., Jestin, A., and Cerutti, M. (1995). Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 gag particles produced in baculovirus-infected cells. J. Virology 69, 4060-4068.

    Nguyen, D. and Hildreth, J. (2000). Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J. Virology 74, 3264-3272.

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