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 Mixlow 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.