WO2010085844A1 - Measuring g protein coupled receptor activation - Google Patents
Measuring g protein coupled receptor activation Download PDFInfo
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- WO2010085844A1 WO2010085844A1 PCT/AU2010/000080 AU2010000080W WO2010085844A1 WO 2010085844 A1 WO2010085844 A1 WO 2010085844A1 AU 2010000080 W AU2010000080 W AU 2010000080W WO 2010085844 A1 WO2010085844 A1 WO 2010085844A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
Definitions
- the present invention relates to methods and polypeptides for detecting a compound in a sample.
- the present invention relates to the use of a cell- free composition comprising at least one G protein coupled receptor embedded in a lipid bilayer which when expressed in a cell the N-terminus of the G protein coupled receptor, or subunits thereof, is outside the cell and the C-terminus is inside the cell, and which is capable of binding the compound.
- the composition also comprises at least one accessory molecule that directly or indirectly binds an intracellular loop and/or the C-terminus of the G protein coupled receptor.
- the G protein coupled receptor, and/or accessory molecule when present, in combination comprise a biolumine scent protein and an acceptor molecule, which enables bioluminescent resonance energy transfer (BRET) to be used to detect the compound binding the receptor.
- BRET bioluminescent resonance energy transfer
- G protein coupled receptors are a family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. GPCRs are found only in eukaryotes including yeast and animals. The ligands that bind and activate GPCRs include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. G protein-coupled receptors are involved in many diseases, and are also the target of therapeutic drugs.
- Vertebrate and C. elegans odorant receptors are members of the G- protein coupled receptor (GPCR) family (Buck et al, 1991; Troemel et al, 1995). GPCRs are characterised by their seven transmembrane spanning domains with ligand binding domains inferred to be on the extracellular side of the membrane and G-protein binding domains on the intracellular side. When a receptor binds the ligand, a conformational change occurs in the receptor allowing it to activate a heterotrimeric G- protein (Kobilka et al., 2007). The activated G-protein can then activate signal transduction pathways such as the guanyl cyclase or phospholipase C pathways, transducing the signal to higher processing centres (Gaillard et al., 2004).
- GPCR G- protein coupled receptor
- RET Forster resonance energy transfer, or simply resonance energy transfer (RET), is the non-radiative transfer of energy from an excited state donor molecule to a ground state acceptor molecule (Ghanouni et al., 2001). Energy transfer efficiency is dependent on the distance between the donor and acceptor, the extent of the spectral overlap and the relative orientation of the acceptor and donor dipoles.
- FRET fluorescence resonance energy transfer
- FRET cyan fluorescent protein
- YFP yellow fluorescent protein
- This FRET system has previously been used to quantify direct ligand binding by a number of GPCRs (Lohse et al., 2003 and 2007; Vilardaga et al., 2003; Rochais et al., 2007; Lisenbee et al., 2007).
- One method for monitoring receptor activation involves dual labelling a single GPCR with CFP and YFP at insertion sites within the third intracellular loop and C- terminus, respectively. Excitation of CFP with light at 436 nm causes CFP emission at 480 nm and FRET to YFP, which emits at 535 nm. The efficiency of FRET varies with the sixth power of the distance between donor and acceptor, providing an exquisitively sensitive indication of conformational changes in the GPCR. This was demonstrated with ⁇ 2AR, parathyroid hormone receptor (PTHR), ⁇ l-AR and secretin receptors in intact cells.
- PTHR parathyroid hormone receptor
- BRET bioluminescence resonance energy transfer
- the donor fluorophore of FRET is replaced with a luciferase and the acceptor can be any suitable fluorophore.
- the use of a luciferase avoids the need for illumination as the addition of a substrate initiates bioluminescent emission and hence, RET.
- BRET GFP
- the BRET system offers superior spectral separation between the donor and acceptor emission peaks of -115 nm compared to ⁇ 55nm for the BRET system at the expense of the quantum yield (Pfleger and Eidne et al., 2006).
- BRET cell-free bioluminescence resonance energy transfer
- GPCRs G protein coupled receptors
- ORs odorant receptors
- the present invention provides a method of detecting a compound, the method comprising, i) contacting a sample with a cell-free composition comprising a) at least one G protein coupled receptor embedded in a lipid bilayer, and which is capable of binding the compound, and b) optionally at least one accessory molecule that directly or indirectly binds an intracellular loop and/or the C-terminus of the G protein coupled receptor, wherein the G protein coupled receptor comprises one or more subunits that are the same or different, and wherein the G protein coupled receptor, and/or accessory molecule when present, in combination comprise a biolurnmeseent protein and an acceptor molecule, ii) simultaneously or sequentially with step i) providing a substrate of the bioiummeseent protein, and allowing the biolun ⁇ nesc ⁇ nt protein to modify the substrate, iii) determining if step ii) modulates bioluminescent resonance energy transfer (BRET) between the bioluminescent protein and the acceptor molecule, where
- BRET biolum
- a subunit of the G protein coupled receptor which is capable of binding the compound comprises i) the bi ⁇ lumin ⁇ tjcent protein, and ii) the acceptor molecule, and wherein the spatial location and/or dipole orientation of the bioluminescent protein relative to the acceptor molecule is altered when the compound binds the subunit.
- the subunit may comprise i) an amino acid sequence provided as SEQ ID NO: 13, 15, 52 or 54, or ii) an amino acid sequence which is at least 40% identical to any one or more of SEQ ID NO: 13, 15, 52 or 54.
- the G protein coupled receptor comprises i) a first subunit comprising a) the bioluminescem protein, and b) the acceptor molecule, and ii) a second subunit which is capable of binding the compound, and wherein the spatial location and/or dipole orientation of the biuluminescent protein relative to the acceptor molecule is altered when the compound binds the second subunit.
- the biolurmnesecnt protein forms part of the third non-transmembrane loop of the subunit, and the acceptor molecule forms part of the fifth non-transmembrane loop
- the acceptor molecule forms part of the third non-transmembrane loop of the subunit
- the biolumincscent protein forms part of the fifth non-transmembrane loop
- the hioluminescent protein forms part of the first non-transmembrane loop of the subunit, and the acceptor molecule forms part of the third non-transmembrane loop
- the acceptor molecule forms part of the first non-transmembrane loop of the subunit, and the biolumitiebcent protein forms part of the third non-transmembrane loop
- the biol ⁇ mincsccnt protein forms part of the fifth non-transmembrane loop of the subunit, and the acceptor molecule forms part of
- the G protein coupled receptor comprises i) a first subunit comprising the bioluminescent protein, and ii) a second subunit comprising the acceptor molecule.
- the first or second subunit may comprise: i) an amino acid sequence provided as SEQ ID NO: 17 or 19, or ii) an amino acid sequence which is at least 40% identical to SEQ ID NO: 17 and/or 19.
- the bioluminebccnt protein forms part of the third non-transmembrane loop of the first subunit, and the acceptor molecule forms part of the fifth non-transmembrane loop of the second subunit, or ii) the acceptor molecule forms part of the third non-transmembrane loop of the first subunit, and the bioluminescent protein forms part of the fifth non-transmembrane loop of the second subunit, or iii) the biolumin ⁇ sceiu protein forms part of the first non-transmembrane loop of the first subunit, and the acceptor molecule forms part of the third non-transmembrane loop of the second subunit, or iv) the acceptor molecule forms part of the first non-transmembrane loop of the first subunit, and the biolumincscent protein forms part of the third non-transmembrane loop of the second subunit, or v) the bioiuminescent protein forms part of the fifth non-transmembrane
- the G protein coupled receptor comprises a subunit comprising the bi ⁇ iuminesccrit protein, and the accessory molecule comprises the acceptor molecule, or ii) the G protein coupled receptor comprises a subunit comprising the acceptor molecule, and the accessory molecule comprises the bi ⁇ luminehccnt protein.
- the accessory molecule comprises i) the bioJumincscent protein, and ii) the acceptor molecule.
- the composition comprises at least two accessory molecules, and wherein a first accessory molecule comprises the biolumineseent protein and a second accessory molecule comprises the acceptor molecule.
- the present inventors were particularly surprised to find how superior the present invention is when compared to FRET-cellfree and BRET -whole cell detection systems.
- the method of the invention is at least 2 fold, 3 fold or 4 fold more sensitive than if a non-biuluminescent protein it> used as ⁇ & donor molecule and a modulation of fluorescence resonance energy transfer (FRET) is determined.
- the method of the invention provides at least a 10 fold, 20 fold, 30 fold or 40 fold more intense BRET signal than if the method used the same G protein coupled receptor present in an intact cell instead of being in a cell-free composition.
- the G protein coupled receptor is a Class A GPCR.
- the class A GPCR is an odorant receptor.
- the odorant receptor can be from any source as long as when expressed in a cell the N-terminus of the receptor is outside the cell and the C-terminus is inside the cell.
- chordate receptors examples include, but are not limited to, mammalian receptors, avian receptors and fish receptors.
- odorant receptor is a nematode receptor or biologically active variant or fragment thereof.
- the nematode receptor is a Caenorhabditis elegans receptor, or biologically active variant or fragment thereof.
- the odorant receptor comprises: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 6, and ii) an amino acid sequence which is at least 40% identical to any one or more of SEQ ID NO: l to 6.
- the subunit is a chimera of a portion of two or more different G protein coupled receptor subunits.
- the accessory molecule can be any molecule that directly or indirectly associates with the G protein coupled receptor. Examples include, but are not limited to, G protein and arrestin.
- the level of BRET is indicative of the relative amount of the compound in the sample.
- bioluminescent proteins include, but are not limited to, a luciferase, a ⁇ -galactosidase, a lactamase, a horseradish peroxidase, an alkaline phophatase, a ⁇ - glucuronidase or a ⁇ -glucosidase.
- luciferases include, but are not limited to, a Renilla luciferase, a Firefly luciferase, a Coelenterate luciferase, a North American glow worm luciferase, a click beetle luciferase, a railroad worm luciferase, a bacterial luciferase, a Gaussia luciferase, Aequorin, a Arachnocampa luciferase, or a biologically active variant or fragment of any one, or chimera of two or more, thereof.
- substrates include, but are not limited to, beetle luciferin, other luciferins, coelenterazine, or a derivative of coelenterazine. Also, in the case of some luminescent proteins such as aequorin, the substrate may be a cofactor such as calcium ions.
- the acceptor molecule is a protein, examples of which include, but are not limited to, green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, t-dimer2, t- dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein or a Phycobiliprotein, or a biologically active variant or fragment of any one thereof.
- GFP green fluorescent protein
- BFP blue fluorescent variant of GFP
- CFP yellow fluorescent variant of GFP
- the acceptor molecule is a non-protein, examples of which include, but are not limited to, an Alexa Fluor dye, Bodipy dye, Cy dye, fluorescein, dansyl, umbelliferone, fluorescent microsphere, luminescent microsphere, fluorescent nanocrystal, Marina Blue, Cascade Blue, Cascade Yellow, Pacific Blue, Oregon Green, Tetramethylrhodamine, Rhodamine, Texas Red, rare earth element chelates, or any combination or derivatives thereof.
- the methods further comprise simultaneously or sequentially with step i) or step ii) providing a co-factor of the biolurmnescenT protein.
- a co-factor of the biolurmnescenT protein examples include, but are not limited to, ATP, magnesium, oxygen, FIvINiH 2 , calcium, or a combination of any two or more thereof,
- the cell-free composition was obtained by producing the G protein coupled receptor in a recombinant cell and disrupting the membrane of the cell.
- the recombinant cell does not produce any non-endogenous proteins which associate with the G protein coupled receptor.
- the cell from which the cell-free composition can be obtained is any cell type capable of expressing the G protein coupled receptor and incorporating the receptor into its cell membrane.
- the recombinant cell is a yeast cell.
- the G protein coupled receptor is embedded in the lipid bilayer of a liposome.
- the method is preformed using microfluidics.
- the present invention provides a purified and/or recombinant polypeptide for detecting a compound, the polypeptide comprising, i) a subunit of a G protein coupled receptor, and ii) a bioluminescent protein and/or an acceptor molecule, wherein when expressed in a cell the N-terminus of the subunit is outside the cell and the C-terminus inside the cell.
- the polypeptide forms part of a protein complex which is a G protein coupled receptor comprising one or more different G protein coupled receptor subunits, and optionally one or more different accessory molecules.
- the biolurmnesecnt protein forms part of the third non-transmembrane loop of the subunit, and the acceptor molecule forms part of the fifth non-transmembrane loop, or ii) the acceptor molecule forms part of the third non-transmembrane loop of the subunit, and the biolumincscent protein forms part of the fifth non-transmembrane loop, or iii) the hioluminescent protein forms part of the first non-transmembrane loop of the subunit, and the acceptor molecule forms part of the third non-transmembrane loop, or iv) the acceptor molecule forms part of the first non-transmembrane loop of the subunit, and the biolumitiescent protein forms part of the third non-transmembrane loop, or v) the biol ⁇ mincscent protein forms part of the fifth non-transmembrane loop of the subunit, and the acceptor molecule forms part of the C-terminus, or vi
- the polypeptide comprises the bioluminescent protein and the acceptor molecule, and wherein the spatial location and/or dipole orientation of the bioluminescent protein relative to the acceptor molecule is altered when the compound binds the polypeptide.
- the polypeptide comprises the subunit and the bioluminescent protein, and the polypeptide is directly or indirectly bound to a second polypeptide comprising an acceptor molecule, and wherein the spatial location and/or dipole orientation of the bioluminescent protein relative to the acceptor molecule is altered when the compound binds the polypeptide and/or the second polypeptide.
- the polypeptide comprises the subunit and the acceptor molecule, and the polypeptide is directly or indirectly bound to a second polypeptide comprising a bioluminescent protein, and wherein the spatial location and/or dipole orientation of the biolumincscent protein relative to the acceptor molecule is altered when the compound binds the polypeptide and/or the second polypeptide.
- the second polypeptide either comprises an G protein coupled receptor subunit (to form a homo- or hetero-dimer or higher multimer as defined herein) or an accessory molecule.
- the present invention provides a purified and/or recombinant polypeptide for detecting a compound, the polypeptide comprising, i) an accessory molecule that directly or indirectly binds an intracellular loop and/or the C-terminus of the G protein coupled receptor, and ii) a bioluminescent protein and/or an acceptor molecule, wherein when expressed in a cell the N-terminus of the G protein coupled receptor, or subunits thereof, is outside the cell and the C-terminus inside the cell.
- polypeptide of the invention Also provided is an isolated and/or exogenous polynucleotide encoding a polypeptide of the invention.
- the polynucleotide comprises: i) a nucleotide sequence as provided in any one of SEQ ID NO's 7 to 12, 14, 16, 18, 20, 51 or 53, or ii) a nucleotide sequence which is at least 40% identical to any one or more of SEQ ID NO's 7 to 12, 14, 16, 18, 20, 51 or 53.
- a vector comprising a polynucleotide of the invention.
- the polynucleotide is operably linked to a promoter.
- a host cell comprising the polynucleotide of the invention and/or the vector of the invention.
- the host cell produces, and hence comprises, a polypeptide of the invention.
- the host cell can be any cell type.
- the present inventors are also the first to show the functional expression of a nematode odorant receptor, which are evolutionarily different to mammalian odorant receptors, in yeast membranes.
- the host cell is a yeast cell.
- the present invention provides a composition comprising the polypeptide of the invention, the polynucleotide of the invention, the vector of the invention, and/or the host cell of the invention.
- the polypeptide is embedded in a lipid bilayer.
- a cell-free composition comprising, i) the polypeptide of the invention comprising a G protein coupled receptor subunit, wherein the polypeptide is embedded in a lipid bilayer, and/or ii) the polypeptide of the invention comprising an accessory molecule which is directly or indirectly bound to an intracellular loop and/or the C-terminus of a G protein coupled receptor, wherein the G protein coupled receptor is embedded in a lipid bilayer, and wherein when expressed in a cell the N-terminus of the G protein coupled receptor, or subunits thereof, is outside the cell and the C-terminus inside the cell.
- the lipid bilayer is a yeast lipid bilayer.
- the cell-free composition further comprises a substrate of the biol ⁇ mincsccnt protein and/or a co-factor of the bioliiminesccnt protein.
- the present invention provides a method of producing a cell- free composition of the invention, the method comprising obtaining a cell of the invention and disrupting the membrane of the cells.
- the cells are permeabilized or lysed.
- the cells are lysed in a French press.
- the present invention provides a biosensor comprising a polypeptide of the invention, a host cell of the invention, a composition of the invention and/or a cell-free composition of the invention.
- the present invention provides a method for screening for a compound that binds a G protein coupled receptor, the method comprising, i) contacting a candidate compound with a cell-free composition comprising a) at least one G protein coupled receptor embedded in a lipid bilayer, and which is capable of binding the compound, and b) optionally at least one accessory molecule that directly or indirectly binds an intracellular loop and/or the C-terminus of the G protein coupled receptor, wherein the G protein coupled receptor comprises one or more subunits that are the same or different, and wherein the G protein coupled receptor, and/or accessory molecule when present, in combination comprise a bi ⁇ luminet>cctH protein and an acceptor molecule, ii) simultaneously or sequentially with step i) providing a substrate of the bioiuminesccnt protein, and allowing the bi ⁇ luminet>cent protein to modify the substrate, iii) determining if step ii) modulates bioluminescent resonance energy transfer (BRET) between the
- the present invention provides a kit comprising a polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, a host cell of the invention, a composition of the invention, a cell-free composition of the invention, and/or a biosensor of the invention.
- Diacetyl binding could result in a conformational change resulting in a decrease (top right), or increase (bottom right), in the distance between RLuc and GFP or a change in the relative orientations of their chromophore dipoles, which would alter
- FIG. 7 Spectral scans of OCOY upon the addition of different concentrations of diacetyl. Top: Raw data. Bottom: Normalized (500 nm) data.
- FIG. 1 ODR-IO transduction cassette. Dark - ODR-IO sequence, Light - variable receptor ligand binding domains. A) N-terminal fixed in cassette, B) N-terminal
- FIG. 12 Co-expressed odr-10-Rluc and odr-10-GFP 2 yeast cells to show odr-10 receptor localization and tagged odr-10 expression level by confocal microscopy (Ex488 nm, Leica SP2 confocal laser scanning microscope).
- FIG. 13 Constitutive BRET 2 of nematode odr-10 in living Invsclyeast cells. All tested yeast strains were induced for expressing tagged odr-10 proteins for 24 hours at 15°C. Energy transfer measurements were performed in living cells by adding 10 uM coelenterazine h (DeepBlueC) and measuring light emissions in a dual wavelength microplate reader with Rluc and GFP2 filter settings as described in the method. Data are means + StD of two independent experiments. Insvcl/odr-10-RLuc - negative control. Invscl/odr-10-RLuc + Invscl/odr-10-GFP2 - OR and OG in separate cells. Cells mixed together for plate reading - BRET is not expected because the two constructs were expressed in separate cells.
- Figure 14 Detection of Odr-10 oligomers by immunoprecipitation. Crude membrane from Invscl yeast cells co-expressing the indicated receptors were solubilised and adjusted to the same amount of luciferase activity. Receptors were then immunoprecipitated with anti-GFP antibody and luciferase activity determined in precipitates. Values are presented as % of maximal amount of precipitated luciferase activity. Data are mean of two biological repeats.
- Figure 15 Measured light output in the BRET 2 channels upon the addition of 5 ⁇ M of coelenterazine 400a to 100 ⁇ L of a whole-cell assay sample of 100 ⁇ L of the cell-free membrane preparations following ultracentrifugation.
- SEQ ID NO: 13 Polypeptide of the invention comprising C. elegans OdrlO receptor
- RLuc inserted into the third intracellular loop (fifth non-transmembrane loop) and GFP inserted at the C-terminus.
- SEQ ID NO: 15 Polypeptide of the invention comprising C. elegans OdrlO receptor
- GFP inserted into the third intracellular loop (fifth non-transmembrane loop) and RLuc inserted at the C-terminus.
- SEQ ID NO: 16 Open reading frame encoding polypeptide of the invention comprising C. elegans OdrlO receptor, GFP inserted into the third intracellular loop
- SEQ ID NO: 17 Polypeptide of the invention comprising C. elegans OdrlO receptor and RLuc inserted at the C-terminus.
- SEQ ID NO: 18 Open reading frame encoding polypeptide of the invention comprising C. elegans OdrlO receptor RLuc inserted at the C-terminus.
- SEQ ID NO: 19 Polypeptide of the invention comprising C. elegans OdrlO receptor and GFP2 inserted at the C-terminus.
- SEQ ID NO:20 Open reading frame encoding polypeptide of the invention comprising C. elegans OdrlO receptor GFP2 inserted at the C-terminus.
- SEQ ID NO:48 Open reading frame encoding mCFP derivative.
- SEQ ID NO:51 Open reading frame encoding Strl 12 BRET dual labelled fusion protein.
- SEQ ID NO:53 Open reading frame encoding mouse 0C 2A adrenergic receptor BRET dual labelled fusion protein.
- SEQ ID NO:55 Open reading frame encoding mouse 0C 2A adrenergic receptor.
- G protein coupled receptor refers to a seven transmembrane receptor which signals through G proteins.
- the receptor may be a single subunit, or two or more receptor subunits. When two or more receptor subunits are present they may be the same, different, or a combination thereof (for example, two of one subunit and a single of another subunit).
- G protein coupled receptor and “subunit of a G protein coupled receptor”, or variations thereof, are used interchangeably.
- the term "odorant receptor”, “olfactory receptor”, “OR” or variations thereof refers to a polypeptide which, when present in a cell of an organism, is involved in chemosensory perception. In an embodiment, the cell is a neuron. Furthermore, the term “odorant receptor” or “olfactory receptor” refers to a polypeptide which binds an odorant ligand, or forms part of a protein complex that binds to an odorant ligand, resulting in a physiologic response. As used herein, the term “bioluminescent protein” refers to any protein capable of acting on a suitable substrate to generate luminescence.
- substrate refers to any molecule that can be used in conjunction with a bioluminescent protein to generate or absorb luminescence.
- the phrase "allowing the bioiumincscent protein to modify the substrate” refer?, to any enzymatic activity of the bioluminescent protein on the substrate that produces energy.
- acceptor molecule refers to any compound which can accept energy emitted as a result of the activity of a bioluminescent protein, and re-emit it as light energy.
- bioluminescent resonance energy transfer is a proximity assay based on the non-radioactive transfer of energy between the bi ⁇ iuminet>cent protein donor and the acceptor molecule.
- modulate or “modulation” or variations thereof refer to an alteration in the intensity and/or emission spectra of the bioluminescent protein and/ or acceptor molecule.
- spatial location refers to the three dimensional positioning of the bioluminescent protein relative to the acceptor molecule which changes as a result of the compound binding a polypeptide defined herein comprising a G protein coupled receptor.
- dipole orientation refers to the direction in three- dimensional space of the dipole moment associated either with the bioluminescent protein and/or the acceptor molecule relative their orientation in three-dimensional space.
- the dipole moment is a consequence of a variation in electrical charge over a molecule.
- the term "more sensitive” refers to a greater change in resonance energy transfer ratio between the ligand unbound form to the ligand bound form of one reporter system (for example, BRET) to another reporter system (for example, FRET).
- BRET reporter system
- FRET reporter system
- As used h containing an isolated and/or recombinant polypeptide of the invention comprising a G protein coupled receptor subunit.
- the cell-free composition comprises no live cells, whether they comprise a polypeptide of the invention or not.
- the term "contacting" refers t ⁇ the addition of a sample comprising, or which may comprise, tbe compound to be delected in a way that the compound is capable of binding the G protein coupled receptor.
- lipid bilayer refers to two layers of, typically amphiphilic, molecular arrays arranged opposite to each other with a common hydrophobic bilayer interior and two hydrophilic surfaces.
- the lipid bilayer can be naturally occurring or artificial.
- the lipid bilayer is a cellular or bio- membrane into which a polypeptide defined herein comprising an G protein coupled receptor is inserted, for example a mammalian, insect, plant or yeast cell membrane, most preferably of a yeast cell membrane.
- yeast lipid bilayer refers to the lipid bilayer being derived from a yeast cell expressing a polypeptide defined herein comprising an G protein coupled receptor.
- the skilled person can readily determine if a lipid bilayer is derived from a yeast by detecting proteins naturally occurring in yeast which are embedded in the membrane such as, but not limited to, Fuslp protein (Trueheart and Fink, 1989), SNARE complex which comprises the Ssol/2p, Sncip and Sec9p proteins (Strop et al., 2007), pheromone receptor Ste2p (Celic et al, 2003), and/or Alrl (Graschopf et al., 2001).
- the term "simultaneously or sequentially" means that the substrate can be provided before, during or after the sample is contacted with a polypeptide as defined herein. Preferably, the substrate is provided at the same time or after the sample.
- sample can be any substance or composition suspected of comprising a compound to be detected.
- samples include air, liquid, biological material and soil.
- the sample may be obtained directly from the environment or source, or may be at least partially purified by a suitable procedure before a method of the invention is performed.
- G protein coupled receptor, and/or accessory molecule when present, in combination comprise a biolumincscent protein and an acceptor molecule means that the bioiuminesccnt protein it> associated, preferably covalently attached, more preferably produced as a fusion protein, with a siibimit of the G protein coupled receptor or with an accessory molecule, and the acceptor molecule is associated, preferably covalemly attached, more preferably produced as a fusion protein, with a subnnit of the G protein coupled receptor or with an accessory molecule.
- the biolwninesccm protein and accessory molecule may be associated, preferably covalently attached, more preferably produced as a fusion protein, with the same or different s ⁇ bumts of the G protein coupled receptor or the same or different accessory molecules.
- one of the biolumincscent protein and accessory molecule may be associated, preferably covalently attached, more preferably produced as a fusion protein, with a bubunit of the G protein coupled receptor, and the other of the bioiurmnescent protein and accessory molecule may be associated with an accessory molecule.
- multiple biolumincscent protein and accessory molecule pairs may be present in the combination for multiplexing to detect different ligands, or increase the sensitivity of the detection of the same ligand.
- the bioluminescent protein or acceptor molecule will not be inserted such that it makes the G protein coupled receptor portion of a polypeptide of the invention incapable of binding the target compound to result in a spatial change to the location and/or dipole orientation of the bioluminescent protein relative to the acceptor molecule.
- the term "at least a majority" of a specified portion (domain) of a G protein coupled receptor refers to at least 51%, more preferably at least 75% and even more preferably at least 90% of the specified region.
- substantially purified or “purified” we mean a polypeptide that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. However, at present there is no evidence that the polypeptides of the invention exist in nature.
- the term "recombinant" in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state.
- the cell is a cell that does not naturally produce the polypeptide.
- the cell may be a cell which comprises a non-endogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced.
- a recombinant polypeptide of the invention includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
- polypeptide and protein are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors.
- proteins and polypeptides as used herein also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides described herein.
- the query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
- a "biologically active fragment” is a portion of a polypeptide as described herein which maintains a defined activity of the full-length polypeptide.
- a biologically active fragment of a G protein coupled receptor must be capable of binding the target compound resulting in a conformational change.
- Biologically active fragments can be any size as long as they maintain the defined activity.
- biologically active fragments are at least 150, more preferably at least 250 amino acids in length.
- a "biologically active variant” is a molecule which differs from a naturally occurring and/or defined molecule by one or more amino acids but maintains a defined activity, such as defined above for biologically active fragments.
- Biologically active variants are typically least 50%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, and even more preferably at least 99% identical to the naturally occurring and/or defined molecule.
- the polypeptide or polynucleotide comprises an amino acid sequence which is at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably
- an “isolated polynucleotide”, including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state.
- the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
- exogenous in the context of a polynucleotide refers to the polynucleotide when present in a cell, or in a cell-free expression system, in an altered amount compared to its native state.
- the cell is a cell that does not naturally comprise the polynucleotide.
- the cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded polypeptide.
- An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
- polynucleotide is used interchangeably herein with the term “nucleic acid”.
- Polynucleotides of the present invention may possess, when compared to molecules provided herewith, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues, or modified residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid). Usually, monomers of a polynucleotide are linked by phosphodiester bonds or analogs thereof.
- Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate and phosphoramidate.
- compositions of the present invention may include an "acceptable carrier".
- acceptable carriers include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
- Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
- the exact nature of the "acceptable carrier” will depend on the use of the composition. Considering the uses described herein, and the nature of the component of the invention in the composition, the skilled person can readily determine suitable a "acceptable carrier(s)" for a particular use.
- a bioluminescent protein is an enzyme which converts a substrate into an activated product which then releases energy as it relaxes.
- the activated product (generated by the activity of the bioluminescent protein on the substrate) is the source of the bioluminescent protein-generated luminescence that is transferred to the acceptor molecule.
- bioluminescent proteins there are a number of different bioluminescent proteins that can be employed in this invention (see, for example, Table 1). Given the size of biolumincsceni proteins it was surprising that functional polypeptides useful for the present invention could be produced.
- Light-emitting systems have been known and isolated from many luminescent organisms including bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly click beetles of genus Pyrophorus and the fireflies of the genera Photinus, Photuris, and Luciola. Additional organisms displaying bioluminescence are listed in WO 00/024878, WO 99/049019 and Viviani (2002).
- luciferases which catalyze an energy-yielding chemical reaction in which a specific biochemical substance, a luciferin (a naturally occurring fluorophore), is oxidized by an enzyme having a luciferase activity (Hastings, 1996).
- a luciferin a naturally occurring fluorophore
- eukaryotic including species of bacteria, algae, fungi, insects, fish and other marine forms can emit light energy in this manner and each has specific luciferase activities and luciferins which are chemically distinct from those of other organisms.
- Luciferin/luciferase systems are very diverse in form, chemistry and function.
- Bioluminescent proteins with luciferase activity are thus available from a variety of sources or by a variety of means. Examples of bioluminescent proteins with luciferase activity may be found in US 5,229,285, 5,219,737, 5,843,746, 5,196,524, and 5,670,356. Two of the most widely used luciferases are: (i) Renilla luciferase (from R.
- reniformis a 35 kDa protein, which uses coelenterazine as a substrate and emits light at 480 nm (Lorenz et al., 1991); and (ii) Firefly luciferase (from Photinus pyralis), a 61 kDa protein, which uses luciferin as a substrate and emits light at 560 nm (de Wet et al., 1987).
- Gaussia luciferase (from Gaussia princeps) has been used in biochemical assays (Verhaegen et al., 2002). Gaussia luciferase is a 20 kDa protein that oxidises coelenterazine in a rapid reaction resulting in a bright light emission at 470 nm.
- Luciferases useful for the present invention have also been characterized from Anachnocampa sp (WO 2007/019634). These enzymes are about 59 kDa in size and are ATP-dependent luciferases that catalyze luminescence reactions with emission spectra within the blue portion of the spectrum.
- non-luciferase, bioluminescent proteins that can be employed in this invention are any enzymes which can act on suitable substrates to generate a luminescent signal.
- enzymes include ⁇ -galactosidase, lactamase, horseradish peroxidase, alkaline phophatase, ⁇ -glucuronidase and ⁇ -glucosidase.
- Synthetic luminescent substrates for these enzymes are well known in the art and are commercially available from companies, such as Tropix Inc. (Bedford, MA, USA).
- a bioluminescent protein with a small molecular weight is used to prevent an inhibition of the interaction due to steric hindrance.
- the bioluminescent protein preferably consists of a single polypeptide chain. Also the bioluminescent proteins preferably do not form oligomers or aggregates.
- the bioluminescent proteins Renilla luciferase, Gaussia luciferase and Firefly luciferase meet all or most of these criteria.
- the choice of the substrate can impact on the wavelength and the intensity of the light generated by the bioluminescent protein.
- a widely known substrate is coelenterazine which occurs in cnidarians, copepods, chaetgnaths, ctenophores, decapod shrimps, mysid shrimps, radiolarians and some fish taxa (Greer and Szalay, 2002).
- coelenterazine analogues/derivatives are available that result in light emission between 418 and 512 nm (Inouye et al., 1997).
- a coelenterazine analogue/derivative 400A, DeepBlueC
- WO 01/46691 Other examples of coelenterazine analogues/derivatives are EnduRen and ViviRen.
- Luciferin refers to a class of light-emitting biological pigments found in organisms capable of bioluminescence, which are oxidised in the presence of the enzyme luciferase to produce oxyluciferin and energy in the form of light. Luciferin, or 2-(6-hydroxybenzothiazol-2-yl)-2-thiazoline-4-carboxylic acid, was first isolated from the firefly Photinus pyralis.
- luciferin There are at least five general types of luciferin, which are each chemically different and catalysed by chemically and structurally different luciferases that employ a wide range of different cofactors.
- First is firefly luciferin, the substrate of firefly luciferase, which requires ATP for catalysis (EC 1.13.12.7).
- Second is bacterial luciferin, also found in some squid and fish, that consists of a long chain aldehyde and a reduced riboflavin phosphate.
- Bacterial luciferase is FMNH-dependent.
- dinoflagellate luciferin a tetrapyrrolic chlorophyll derivative found in dinoflagellates (marine plankton), the organisms responsible for night-time ocean phosphorescence.
- Dinoflagellate luciferase catalyses the oxidation of dinoflagellate luciferin and consists of three identical and catalytically active domains.
- imidazolopyrazine vargulin which is found in certain ostracods and deep-sea fish, for example, Porichthys.
- Last is coelanterazine (an imidazolpyrazine), the light-emitter of the protein aequorin, found in radiolarians, ctenophores, cnidarians, squid, copepods, chaetognaths, fish and shrimp.
- acceptor molecules may be a protein or non-proteinaceous.
- acceptor molecules that are protein include, but are not limited to, green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), HcRed, t- HcRed, DsRed, DsRed2, t-dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein or a Phycobiliprotein, or a biologically active variant or fragment of any one thereof
- acceptor molecules that are not proteins include, but are not limited to, Alexa Fluor dye, Bodipy dye, Cy dye, fluorescein, dansyl, umbelliferone, fluorescent microsphere, luminescent microsphere, fluorescent nanocrystal, Marina Blue, Cascade Blue, Cascade Yellow, Pacific Blue, Oregon Green, Tetramethylrhodamine, Rhodamine, Texas Red, rare earth element chelates, or any combination or derivatives thereof.
- GFPs green fluorescent protein from the jellyfish Aequorea victoria and numerous other variants (GFPs) arising from the application of molecular biology, for example mutagenesis and chimeric protein technologies (Tsien, 1998).
- GFPs are classified based on the distinctive component of their chromophores, each class having distinct excitation and emission wavelengths: class 1, wild-type mixture of neutral phenol and anionic phenolate: class 2, phenolate anion : class 3, neutral phenol : class 4, phenolate anion with stacked s-electron system: class 5, indole : class 6, imidazole : and class 7, phenyl.
- a naturally occurring acceptor molecule which has been mutated (variants) can also be useful for the present invention.
- One example of an engineered system which is suitable for BRET is a Renilla luciferase and enhanced yellow mutant of GFP (EYFP) pairing which do not directly interact to a significant degree with one another alone in the absence of a mediating protein(s) (in this case, the G protein coupled receptor) (Xu et al., 1999).
- the acceptor molecule is a fluorescent nanocrystal. Nanocrystals, or "quantum dots", have several advantages over organic molecules as fluorescent labels, including resistance to photodegradation, improved brightness, non- toxicity, and size dependent, narrow emission spectra that enables the monitoring of several processes simultaneously. Additionally, the absorption spectrum of nanocrystals is continuous above the first peak, enabling all sizes, and hence all colors, to be excited with a single excitation wavelength.
- Fluorescent nanocrystals may be attached, or "bioconjugated", to proteins in a variety of ways.
- the surface cap of a "quantum dot” may be negatively charged with carboxylate groups from either dihydrolipoic acid (DHLA) or an amphiphilic polymer.
- DHLA dihydrolipoic acid
- Proteins can be conjugated to the DHLA-nanocrystals electrostatically, either directly or via a bridge consisting of a positively charged leucine zipper peptide fused to recombinant protein. The latter binds to a primary antibody with specificity for the intended target.
- antibodies, streptavidin, or other proteins are coupled covalently to the polyacrylate cap of the nanocrystal with conventional carbodiimide chemistry.
- quantum dots can contain as few as 100 to 100,000 atoms within the quantum dot volume, with a diameter of 10 to 50 atoms.
- Some quantum dots are small regions of one material buried in another with a larger band gap.
- These can be so-called core-shell structures, for example, with CdSe in the core and ZnS in the shell or from special forms of silica called ormosil. The larger the dot, the redder (lower energy) its fluorescence spectrum. Conversely, smaller dots emit bluer (higher energy) light.
- the coloration is directly related to the energy levels of the quantum dot.
- the bandgap energy that determines the energy (and hence color) of the fluoresced light is inversely proportional to the square of the size of the quantum dot. Larger quantum dots have more energy levels which are more closely spaced. This allows the quantum dot to absorb photons containing less energy, i.e. those closer to the red end of the spectrum.
- the acceptor molecule is a fluorescent microsphere.
- fluorescent microspheres typically made from polymers, and contain fluorescent molecules (for example fluorescein GFP or YFP) incorporated into the polymer matrix, which can be conjugated to a variety of reagents.
- Fluorescent microspheres may be labelled internally or on the surface. Internal labelling produces very bright and stable particles with typically narrow fluorescent emission spectra. With internal labelling, surface groups remain available for conjugating ligands (for example, proteins) to the surface of the bead. Internally-labelled beads are used extensively in imaging applications, as they display a greater resistance to photobleaching.
- Carboxylate-modified fluorescent microspheres are suitable for covalent coupling of proteins using water-soluble carbodiimide reagents such as l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDAC).
- Sulfate fluorescent microspheres are relatively hydrophobic and will passively and nearly irreversibly adsorb almost any protein.
- Aldehyde-sulfate fluorescent microspheres are sulfate microspheres that have been modified to add surface aldehyde groups, and react with proteins.
- the acceptor molecule is a luminescent microsphere.
- luminescent microsphere typically made from polymers, which contain luminescent molecules (for example complexes of europium or platinum) incorporated into the polymer matrix, which can be conjugated to a variety of reagents.
- a criteria which should be considered in determining suitable pairings for BRET is the relative emission/fluorescence spectrum of the acceptor molecule compared to that of the bioluminescent protein.
- the emission spectrum of the bioluminescent protein should overlap with the absorbance spectrum of the acceptor molecule such that the light energy from the bioluminescent protein luminescence emission is at a wavelength that is able to excite the acceptor molecule and thereby promote acceptor molecule fluorescence when the two molecules are in a proper proximity and orientation with respect to one another.
- Renilla luciferase/EGFP pairing is not as good as an Renilla luciferase/EYEF pairing based on observable emission spectral peaks (Xu, 1999; Wang, et al. (1997) in Bioluminescence and Chemiluminescence : Molecular Reporting with Photons, eds. Hastings et al. (Wiley, New York), pp. 419-422).
- protein fusions are prepared containing the selected bioluminescent protein and acceptor molecule and are tested, in the presence of an appropriate substrate.
- bioluminescent protein and acceptor molecule do not spuriously associate with each other. This can be accomplished by separate co-expression of the bioluminescent protein and acceptor molecule in the same cells and then monitoring the luminescence spectrum in order to determine if BRET occurs. This may be achieved, for example, using the method of Xu et al. (1999).
- the selected bioluminescent protein and acceptor molecule form a suitable BRET pair if little or no BRET is observed.
- the bioluminescent protein emission can be manipulated by modifications to the substrate. In the case of luciferases the substrate is coelenterazine. The rationale behind altering the bioluminescent protein emission is to improve the resolution between donor emission and acceptor emissions.
- the original BRET system uses the Renilla luciferase as donor, EYFP (or Topaz) as the acceptor and coelenterazine h derivative as the substrate. These components when combined in a BRET assay, generate light in the 475-480 nm range for the bioluminescent protein and the 525-530 nm range for the acceptor molecule, giving a spectral resolution of 45-55 nm.
- Renilla luciferase generates a broad emission peak overlapping substantially the GFP emission, which in turn contributes to decrease the signal to noise of the system.
- One BRET system of the present invention using coeWOOa as the Renilla luciferase substrate, provides broad spectral resolution between donor and acceptor emission wavelengths (-105nm). Renilla luciferase with coeWOOa generates light between 390-400 nm and a GFP was prepared which absorbs light in this range and re-emits light at 505-508 nm.
- this BRET system provides an excellent biological tool to monitor small changes in conformation of a polypeptide of the invention. This is a significant improvement over the system described previously using the coelenterazine h derivative and EYFP, which has a wavelength difference between donor and acceptor of approximately 51 nm.
- coelenterazine derivatives are known in the art, including coel400a, that generate light at various wavelengths (distinct from that generated by the wild type coelenterazine) as a result of Renilla luciferase activity.
- coel400a that generate light at various wavelengths (distinct from that generated by the wild type coelenterazine) as a result of Renilla luciferase activity.
- a worker skilled in the art would appreciate that because the light emission peak of the donor has changed, it is necessary to select an acceptor molecule which will absorb light at this wavelength and thereby permit efficient energy transfer. This can be done, for example by altering a GFP class 4 such that it becomes a class 3 or 1 GFP.
- Spectral overlapping between light emission of the donor and the light absorption peak of the acceptor is one condition among others for an efficient energy transfer.
- Class 3 and 1 GFPs are known to absorb light at 400 nm and re-emit between 505-511 nm.
- G protein-coupled receptors are also known as seven transmembrane receptors, 7TM receptors, heptahelical receptors, and G protein linked receptors (GPLR). GPCRs are a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. The ligands that bind and activate these receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. GPCRs are involved in many diseases, but are also the target of around half of all modern medicinal drugs. GPCRs can be grouped into at least 5 classes based on sequence homology and functional similarity:
- Class C metabotropic/pheromone Class D fungal pheromone
- Class A Rhodopsin like receptors include: Amine receptors: Acetylcholine, Alpha Adrenoceptors, Beta Adrenoceptors, Dopamine, Histamine, Serotonin, Octopamine, and Trace amine; Peptide receptors: Angiotensin, Bombesin, Bradykinin, C5a anaphylatoxin, Fmet-leu-phe, APJ like, Interleukin-8, Chemokine receptors (C-C Chemokine, C-X-C Chemokine, BONZO receptors (CXC6R), C-X3-C Chemokine, and XC Chemokine), CCK receptors, Endothelin receptors, Melanocortin receptors, Neuropeptide Y receptors, Neurotensin receptors, Opioid receptors, Somatostatin receptors, Tachykinin receptors, (Substance P (NKl), Substance K (NK2), Neuromedin K (NK3)
- Class B the secretin-receptor family of the GPCRs includes receptors for polypeptide hormones (Calcitonin, Corticotropin releasing factor, Gastric inhibitory peptide, Glucagon, Glucagon-like peptide- 1,-2, Growth hormone-releasing hormone, Parathyroid hormone, PACAP, Secretin, Vasoactive intestinal polypeptide, Diuretic hormone, EMRl, Latrophilin), molecules thought to mediate intercellular interactions at the plasma membrane (Brain-specific angiogenesis inhibitor (BAI)) and a group of Drosophila proteins (Methuselah-like proteins) that regulate stress responses and longevity.
- polypeptide hormones Calcitonin, Corticotropin releasing factor, Gastric inhibitory peptide, Glucagon, Glucagon-like peptide- 1,-2, Growth hormone-releasing hormone, Parathyroid hormone, PACAP, Secretin, Vasoactive intestinal polypeptide, Diuretic hormone, EMRl, Latrophilin
- BAI Brain-specific angiogenesis inhibitor
- Metabotropic glutamate/pheromone receptors include Metabotropic glutamate, Metabotropic glutamate group I, Metabotropic glutamate group II, Metabotropic glutamate group III, Metabotropic glutamate other, Extracellular calcium-sensing, Putative pheromone Receptors, GABA-B, GABA-B subtype 1, GABA-B subtype 2, and Orphan GPRC5 receptors.
- GPCRs are involved in a wide variety of physiological processes, including the visual sense, the sense of smell, behavioral and mood regulation, regulation of immune system activity and inflammation, autonomic nervous system transmission, cell density sensing, and many others. It is known that the inactive G protein is bound to the receptor in its inactive state. Once the ligand is recognized, the receptor or a subunit thereof shifts conformation and thus mechanically activates the G protein, which detaches from the receptor. The receptor can now either activate another G protein, or switch back to its inactive state. It is believed that a receptor molecule exists in a conformational equilibrium between active and inactive biophysical states. The binding of ligands to the receptor may shift the equilibrium toward the active receptor states.
- Polypeptides of the invention comprise G protein coupled receptors which, when expressed in a cell the N-terminus of the receptor is outside the cell and the C- terminus is inside the cell.
- suitable techniques for detecting the orientation of a transmembrane protein comprise but are not limited to crystallography, >JMR-stuJies, modeling studies as well as microscopy techniques, like imrmmolabcling combined with detergent pcrmeabilisation controls fur light or electron microscopy preparation, t ragmen t complementation tagging of two polypeptides and the like.
- the G protein coupled receptor is a Class A GPCR.
- the class A (rhodopsin-like) GPCR is an odorant receptor or an adrenergic receptor, more preferably an odorant receptor.
- the odorant receptor can be from any source as long as when expressed in a cell the N-terminus of the receptor is outside the cell and the C-terminus is inside the cell. Examples include, but are not limited to, a chordate receptor, a nematode receptor, or a biologically active variant or fragment of any one thereof. Examples of chordate receptors include, but are not limited to mammalian receptors, avian receptors and fish receptors.
- the odorant receptor is a nematode receptor or biologically active variant or fragment thereof.
- the nematode receptor is a Caenorhabditis elegans receptor, or biologically active variant or fragment thereof.
- Examples of odorant receptors that can be used to produce polypeptides of the invention and/or used in the methods of the invention are described in Buck and A' ⁇ ei (1991), Robertson 0 oo 8 and 2001 ). Aior ⁇ et al. (2006), Feldmesser (200ft), Olcnder ct al. (2004a and b), GkiMnan ct zl.
- the OPCR is a (Mass B ⁇ r Class € receptor, with Class C being more preferred of these two embodiments.
- the G protein coupled receptor comprises seven transmembrane domains.
- T he bioluminescent protein can form part of the first, third, fifth non- transmembrane loops (domains) or the C-terminus of the G protein coupled receptor (or polypeptide of the invention).
- the acceptor molecule also can form part of the first, third, fifth non-transmembrane loops (domains) or the C-terminus of the G protein coupled receptor (or polypeptide of the invention). Each of these regions is intracellular when the G protein coupled receptor is expressed and present in a cell.
- the acceptor molecule cannot be in the same region as the bioluminescent protein when part of the same molecule (namely, the same single polypeptide chain), however, the acceptor molecule can be in the equivalent region as the bioluminescent protein when the G protein coupled receptor is present as a dimer or higher multimer.
- the bioluminescent protein can form part of the C-terminus of one subunit of the receptor, and the acceptor molecule can form part of the C-terminus of another subunit of the receptor.
- the subunit to which the label is associated can be the same or different, for instance the two subunits can be identical apart from one labelled with the bioluminescent protein and the other labelled with the acceptor molecule.
- the biorummcscent protein forms part of the third non-transmembrane loop of the GPCR subunit, and the acceptor molecule forms part of the fifth non-transmembrane loop.
- the acceptor molecule forms part of the third non-transmembrane loop of the GPCR subunit, and the biolnmincscent protein forms part of the fifth non-transmembrane loop.
- the bioluminescent protein forms part of the first non- transmembrane loop of the GPCR subunit, and the acceptor molecule forms part of the third non-transmembrane loop.
- the acceptor molecule forms part of the first non-transmembrane loop of the GPCR subunit, and the biolumincscent protein forms part of the third non-transmembrane loop.
- the hioginaneseent protein forms part of the fifth non-transmembrane loop of the GPCR subunit, and the acceptor molecule forms part of the C-terminus.
- the acceptor molecule forms part of the fifth non-transmembrane loop of the GPCR subunit, and the hioluminesc ⁇ nt protein forms part of the C-terminus.
- the G protein coupled receptor comprises at least two subunits, where the bioluminescent protein forms part of the third non-transmembrane loop of a first subunit, and the acceptor molecule forms part of the fifth non- transmembrane loop of a second subunit.
- the acceptor molecule forms part of the third non-transmembrane loop of a first subunit, and the bioluminescent protein forms part of the fifth non-transmembrane loop of a second subunit.
- the G protein coupled receptor comprises at least two subunits, where the hiohimmcseent protein forms part of the first non-transmembrane loop of a first subunit, and the acceptor molecule forms part of the third non- transmembrane loop of a second subunit.
- the acceptor molecule forms part of the first non-transmembrane loop of a first subunit
- the biohiminescent protein forms part of the third non-transmembrane loop of a second subunit.
- the G protein coupled receptor comprises at least two subunits, where the bioluminescent protein forms part of the fifth non-transmembrane loop of a first subunit, and the acceptor molecule forms part of the C-terminus of a second subunit.
- the acceptor molecule forms part of the fifth non-transmembrane loop of a first subunit, and the biolumincscent protein forms part of the C-terminus of a second subunit.
- the G protein coupled receptor comprises at least two subunits and the donor and acceptor molecule are in the same site of the first and second subunits respectively.
- the bioluminescent protein or acceptor molecule is located after the second amino acid of the fifth transmembrane domain and before the second amino acid before the beginning of ⁇ slh transmembrane domain. In another embodiment, the bioluminescent protein or acceptor molecule is located after about amino acid 8 after the fifth transmembrane domain or after about amino acid 22 after the filth transmembrane domain. In a further embodiment, the bioluminescent protein or acceptor molecule is inserted about IU or ⁇ ?. amino acids before the sixth transmembrane domain. Most preferably, the bioluminescent protein or acceptor molecule is located in the middle of the third non -transmembrane loop (domain).
- the bioluminescent protein or acceptor molecule is inserted after abo ⁇ t the 16 or 20 amino acids after the seventh transmembrane.
- bioluminescent protein or acceptor molecule in the first non-transmcmbrane loop (domain), it is preferred that said label is inserted about two amino acids after the end o( first transmembrane domain and about two amino acids before the beginning of the second transmembrane domain. Most preferably, the bioluminescent protein or acceptor molecule is located in tbc middle of the first non- transtnembrane loop (domain).
- the bioluminescent protein can form part of the N- terminus, second, fourth, or sixth non-transmembrane loops (domains) of the G protein coupled receptor (or polypeptide of the invention).
- the acceptor molecule also can form part of the N-terminus, second, fourth, or sixth non-transmembrane loops (domains) of the G protein coupled receptor (or polypeptide of the invention), however, it cannot be in the same region as the bioluminescent protein when part of the same molecule. Each of these regions is extracellular when the G protein coupled receptor is expressed and present in a cell.
- I he GPCR may be a non -naturally occurring chimera of two or more different GPCRs.
- this enables a transduction cassette to be produced where portions of one receptor are always present in the chimera into which other portions of a wide variety of GPCRs are inserted depending on the compound to be detected.
- the subunit comprises the N-terminus and at least a majority of the first transmembrane domain of a first G protein coupled receptor subunit, at least a majority of the first non-transmembrane loop through to at least a majority of the fifth transmembrane domain of a second G protein coupled receptor subunit, and at least a majority of the fifth non-transmembrane loop through to the C-terminal end of the first G protein coupled receptor subunit.
- the subunit comprises the N-terminus through to at least a majority of the fifth transmembrane domain of a first G protein coupled receptor subunit, and at least a majority of the fifth non-transmembrane loop through to the C- terminal end of a second G protein coupled receptor subunit.
- Fhc skilled person cars readily determine the N-te ⁇ sinal end, transmembrane domains, non-transmembrane loops ( domains) and C-terminus of a G protein coupled.
- a variety of hioinformatics approaches may be used to determine tbe location and topology of transmembrane domains in a protein, based on its amino acid sequence and similarity with known transmembrane domain of Cs protein coupled receptor. Alignments and amino acid sequence comparisons are routinely performed in the an, for example, by using the BLAST program or the CLCSTAL W program. Based on alignments with known transmembrane domain-containing proteins, it is possible for one skilled in the art to predict the location of transmembrane domains.
- the 3 dimensional structures of some membrane-spanning proteins are known, for example, the seven transmembrane G-pr ⁇ tein coupled rhodopsm photoreceptor structure has been soiled by x-ray crystallography. Based on analyses and comparisons with such 3D structures, it may be possible to predict the location and topology of transmembrane domains in othef membrane pjoteins. There are also many programs available lor predicting the location and topology of transmembrane domains in proteins.
- TMpred f ⁇ Totmann and Stoffel, 1993 S which predicts membrane spanning proteins segments: TopPred (von Heijnc ct al, 19 1 O) which predicts the topology of membrane proteins; PRFDATOR (Frishrnan and Argos, I 1 W i, which predicts secondary structure from single and multiple sequences; TMAP (Persson and Argos, 1994), which predicts transmembrane regions of proteins from multiply aligned sequences; and ALOV! 2 (KJien et al, 1984), which pf edicts transmembrane regions from single sequences.
- TopPred von Heijnc ct al, 19 1 O
- PRFDATOR Frishrnan and Argos, I 1 W i, which predicts secondary structure from single and multiple sequences
- TMAP Persson and Argos, 1994
- ALOV! 2 KJien et al, 1984
- Amino acid sequence mutants/variants of naturally occurring G protein coupled receptors can be prepared by introducing appropriate nucleotide changes into the encoding polynucleotide, or by in vitro synthesis of the desired polypeptide.
- Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.
- a combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
- Mutant (variant) polypeptides can be prepared using any technique known in the art.
- a polynucleotide described herein can be subjected to in vitro mutagenesis.
- in vitro mutagenesis techniques may include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL- 1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations.
- the polynucleotides encoding G protein coupled receptors are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/variant DNA can readily be screened using techniques described herein to determine if they are useful for the methods of the invention.
- the location of the mutation site and the nature of the mutation will depend on characteristic (s) to be modified.
- the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
- Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
- Substitution mutants have at least one amino acid residue in the G protein coupled receptor removed and a different residue inserted in its place.
- the sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 3.
- unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into a polypeptide described herein.
- Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, 2- aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ - methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids
- polypeptides which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide. able 3. Exemplary substitutions.
- polypeptides described herein can be produced in a variety of ways, including production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides.
- an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide.
- a preferred cell to culture is a recombinant cell of the present invention.
- Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production.
- An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention.
- Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
- Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
- An accessory molecule is a protein which is involved in G coupled protein receptor signalling. Examples include G proteins and arrestins. Any labelled accessory molecule can be used in the methods of the present invention as long as they directly or indirectly associate with the G protein coupled receptor (and/or polypeptide defined herein) and following binding of the compound to the G protein coupled receptor there is a spatial alteration in the relative position of the two labels to allow for BRET detection.
- mutants (variants) described above for G protein coupled receptors also apply to the accessory molecules.
- the bioluminescent protein or acceptor molecule can be located anywhere in the accessory molecule as long as the accessory molecule is still able to directly or indirectly bind the receptor, and following binding of a compound of interest a change in BRET can be detected.
- the bioluminescent protein or acceptor molecule is located at the C-terminus or N-terminus of the accessory molecule.
- the accessory molecule indirectly binds the receptor if it requires at least one other accessory molecule to form a complex with the GPCR, for instance a first accessory molecule binds the receptor and a second accessory molecule indirectly binds the receptor by directly binding to the first accessory molecule.
- G-proteins are a family of proteins involved in second messenger cascades for intracellular signaling. G proteins function as "molecular switches,” alternating between an inactive guanosine diphosphate (GDP) bound state and an active guanosine triphosphate (GTP) bound state. Ultimately, G proteins regulate downstream cell processes by initiating cascades of signal transduction networks Hofmann et al., 2009; Oldham and Hamm, 2008).
- G proteins There are two distinct families of G proteins: Heterotrimeric G proteins, sometimes referred to as the "large” G proteins, that are activated by G protein-coupled receptors and made up of alpha ( ⁇ ), beta ( ⁇ ), and gamma ( ⁇ ) subunits; and "small” G proteins (20-25kDa) that belong to the Ras superfamily of small GTPases. These proteins are homologous to the alpha ( ⁇ ) subunit found in heterotrimeric G proteins, and also bind GTP and GDP and are involved in signal transduction.
- G proteins are covalently modified with lipids, for example heterotrimeric G protein subunits may be myristolated, palmitoylated, or prenylated, while small G proteins may be prenylated.
- G-proteins there are many known G-proteins.
- a number of heterotrimeric G proteins have been identified in nematodes, for example C. elegans has 21 Ga, 2 G ⁇ and 2 G ⁇ genes (Jansen et al., 1999; Cuppen et al., 2003). Based on sequence similarity, mammalian Ga subunits have been divided into four families: Gs, Gi/o, Gq and G12 (Neves et al., 2002).
- C. elegans expresses one ortholog of each of the mammalian families: GSA-I (Gs), GOA-I (Gi/o), EGL-30 (Gq) and GPA- 12 (G 12). The remaining C.
- GPA-1-11, GPA- 13-17 and ODR-3 do not share sufficient homology to allow classification.
- Arrestins are one of the key proteins for the termination of G protein signaling. Activated GPCRs are specifically phosphorylated by G protein-coupled receptor kinases (GRKs) and then bind to arrestins to preclude the receptor/G protein interaction, resulting in quenching of the following signal transduction. Arrestins bind GPCRs, including those that have been agonist-activated and bind more tightly to those that have been phosphorylated by GRKs than those that are not.
- GPKs G protein-coupled receptor kinases
- R-arrestin-binding leads to the uncoupling of the receptor from its cognate G- proteins, causing dampening or desensitization of GPCR signaling via the downstream second messenger molecules.
- arrestin binding can initiate new signaling from GPCRs.
- R-arrestins serve as adaptors, which bring non-receptor tyrosine kinases, such as Src, to form signaling complexes with the internalizing receptor.
- H-arrestins function as GPCR-regulated scaffolds for MAPK modules such as ASK-MKK4-JNK3 and RAF-MEK- ERK 1/2.
- arrestins interact with proteins of the endocytic machinery, such as clathrin, ⁇ -adaptin subunit2 of the AP2 complex, and Arf-6 and thus promote internalization of receptors via clathrin-coated vesicles.
- ARR-I An example of a nematode arrestin is ARR-I from C. elegans, which is primarily expressed in the nervous system, including the HSN neuron and various chemosensory neurons, involved in detecting soluble and volatile odorants.
- ARR-I An example of a nematode arrestin is ARR-I from C. elegans, which is primarily expressed in the nervous system, including the HSN neuron and various chemosensory neurons, involved in detecting soluble and volatile odorants.
- Over 20 putative arrestins have now been identified in the nematode C. elegans. Such arrestins are expressed throughout the nervous system and support receptor internalization, similarly to the vertebrate non-visual arrestins, yet participate in olfaction and vision, similarly to the visual/sensory subtypes.
- One embodiment of the present invention includes a recombinant vector, which comprises at least one isolated/exogenous polynucleotide encoding a polypeptide as described herein, inserted into any vector capable of delivering the polynucleotide molecule into a host cell.
- a vector contains heterologous polynucleotide sequences, that are polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived.
- the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid.
- One type of recombinant vector comprises the polynucleotide(s) operably linked to an expression vector.
- the phrase operably linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
- an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule.
- the expression vector is also capable of replicating within the host cell.
- Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
- Expression vectors include any vectors that function (i.e., direct gene expression) in recombinant cells, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells.
- Vectors of the invention can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.
- operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence.
- a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system.
- promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are c ⁇ -acting.
- some transcriptional regulatory elements, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
- expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention.
- recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
- Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or animal cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SPOl, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcom
- Another embodiment of the present invention includes a host cell transformed with one or more recombinant molecules described herein or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
- Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
- Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention.
- Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides described herein or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule as described herein.
- Host cells of the present invention can be any cell capable of producing at least one protein defined herein, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, animal and plant cells.
- host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-I cells, COS (e.g., COS-7) cells, and Vero cells.
- E. coli including E. coli K- 12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
- yeast cells such as, but not limited to, Saccharomyces spp., in particular, Saccharomyces cerevisiae, Candida spp., Hansenula spp. or Pichia spp.
- Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
- Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
- transcription control signals e.g., promoters, operators, enhancers
- translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
- Methods tor preparing cell-free compositions from cells include repeated cycles of freezing and thawing, grinding, treatment of cells with ultrasound in a sonicator device, homogenization, the use of a French press, the addition of detergent and/or enzymes, glass-bead lysis, differential ccntritugation, several density gradient procedures using a variety of gradient media. These techniques are familiar to the skilled worker and are dealt with in detail for example in "Current Protocols in Protein Science"; John E. Caligan; Ben M. Dunn; Hidde L. Ploegh; David W. Speicher; Paul T. Wingfield; Wiley and Sons).
- Jlicrc are also many commercial kit*-, available for purifying cell membrane fractions, for example, Qiagen's Qpjoteome Plasma ⁇ 'lembrane Protein Kit.
- Cells ate incubated in a hypotonic b ⁇ fier, causing them to swell. A mild detergent is added and the resulting cell suspension is homogenized by mechanical disruption. Intact cells, cell debris, nuclei and major organelles are removed by centrifugation. The resulting supernatant contains eytosolic proteins and microsomes, Golgi vesicles, and plasma membranes. A ligand specific for molecules on the cell membrane is added to the supernatant.
- the ligand binds to the ceil membrane vesicles and the iigand vesicle complexes are precipitated using magnetic beads that bind to the ligand. After washing, plasma membrane vesicles are el ⁇ ted under native conditions and the ligand remains bound to the beads.
- a commonly used method lor preparing ceil membranes is to spher ⁇ plast the cells. This gives high yields, and is ideally suited to the large-scale isolation of plasma membranes.
- Such a method generally entails coating the negatively charged surfaces of the spberoplasts with dense eationie silica beads, which makes the membrane denser than any other membranous organelle of the cell. ⁇ washing procedure removes the excess eationie beads, followed by addition of polyaerylic acid to block the fiee eationie groups on the beads.
- the coated spheroplasts are subsequently lysed by hand homogenizaiion in an _h G I ⁇ - containing lysis butter to prevent aggregation of membrane components.
- Ccrr ⁇ rifugation of the spheropiast lysate pellets the heavy plasma membiane-microbead assemblies, leaving intracellular membranous organelles in the supernatant.
- a polypeptide of the invention comprising a GPCR are embedded in the lipid bilayer of a liposome preparation.
- the term 'liposome' refers to a closed vesicle comprising bilayers of amphiphilic phospholipids, for example phosphatidyl ethanoiamine and cholesterol.
- Liposomes can be composed of naturally-derived phospholipids with mixed lipid chains (like phosphatidylethanolamine), or of pure surfactant components like DOPE (dioleoylphosphatidylethanolamine).
- Liposomes containing polypeptides described herein may be created by disrupting the phospholipid membrane of cells expressing the protein in water, for example by sonication. The phospholipids would reassemble into liposomal spheres containing a core of aqueous solution. Low shear rates would create multilamellar liposomes, which have many layers. Continued high-shear sonication would form smaller unilamellar liposomes, more suited to the application of the present invention. Experimentation to produce the optimal liposome size with optimal orientation and accessibility is within the routine capabilities of one of ordinary skill in the art.
- Polypeptides described herein may also be produced and incorporated into liposomes by membrane reconstitution techniques involving dissociation in detergents, followed by their reconstitution in the presence o( lipids and removal of the detergent.
- the liposome composition would be adjusted so as ro ensure optimal orientation and accessibility of the polypeptides contained in the liposomes with binding partners (compounds). Such adjustments to achieve optima! liposome compositions are routine in the art.
- the energy transfer occurring between the bioluminescent protein and acceptor molecule is presented as calculated ratios from the emissions measured using optical filters (one for the acceptor molecule emission and the other for the bioluminescent protein emission) that select specific wavelengths (see equation 1).
- Ea/Ed BRET ratio (1) where Ea is defined as the acceptor molecule emission intensity (emission light is selected using a specific filter adapted for the emission of the acceptor) and Ed is defined as the bioluminescent protein emission intensity (emission light is selected using a specific filter adapted for the emission of the bioluminescent protein).
- optical filters may be any type of filter that permits wavelength discrimination suitable for BRET.
- optical filters used in accordance with the present invention can be interference filters, long pass filters, short pass filters, etc.
- Intensities (usually in counts per second (CPS) or relative luminescence units (RLU)) of the wavelengths passing through filters can be quantified using either a photo-multiplier tube (PMT) or a CCD camera.
- the quantified signals are subsequently used to calculate BRET ratios and represent energy transfer efficiency.
- the BRET ratio increases with increasing intensity of the acceptor emission.
- a ratio of the acceptor emission intensity over the donor emission intensity is determined (see equation 1), which is a number expressed in arbitrary units that reflects energy transfer efficiency. The ratio increases with an increase of energy transfer efficiency (see Xu et al, 1999).
- Energy transfer efficiencies can also be represented using the inverse ratio of donor emission intensity over acceptor emission intensity (see equation T). In this case, ratios decrease with increasing energy transfer efficiency.
- the emission intensities Prior to performing this calculation the emission intensities are corrected for the presence of background light and auto-luminescence of the substrate. This correction is generally made by subtracting the emission intensity, measured at the appropriate wavelength, from a control sample containing the substrate but no bioluminescent protein, acceptor molecule or polypeptide of the invention.
- Ed/Ea BRET ratio (2) where Ea and Ed are as defined above.
- the light intensity of the bioluminescent protein and acceptor molecule emission can also be quantified using a monochromator-based instrument such as a spectrofluorometer, a charged coupled device (CCD) camera or a diode array detector.
- a monochromator-based instrument such as a spectrofluorometer, a charged coupled device (CCD) camera or a diode array detector.
- CCD charged coupled device
- the emission scan is performed such that both bioluminescent protein and acceptor molecule emission peaks are detected upon addition of the substrate.
- the areas under the peaks represent the relative light intensities and are used to calculate the ratios, as outlined above.
- Any instrument capable of measuring lights for the bioluminescent protein and acceptor molecule from the same sample can be used to monitor the BRET system of the present invention.
- the acceptor molecule emission alone is suitable for effective detection and/or quantification of BRET.
- the energy transfer efficiency is represented using only the acceptor emission intensity. It would be readily apparent to one skilled in the art that in order to measure energy transfer, one can use the acceptor emission intensity without making any ratio calculation. This is due to the fact that ideally the acceptor molecule will emit light only if it absorbs the light transferred from the bioluminescent protein. In this case only one light filter is necessary.
- the bioluminescent protein emission alone is suitable for effective detection and/or quantification of BRET.
- the energy transfer efficiency is calculated using only the bioluminescent protein emission intensity. It would be readily apparent to one skilled in the art that in order to measure energy transfer, one can use the donor emission intensity without making any ratio calculation. This is due to the fact that as the acceptor molecule absorbs the light transferred from the bioluminescent protein there is a corresponding decrease in detectable emission from the bioluminescent protein. In this case only one light filter is necessary.
- the energy transfer efficiency is represented using a ratiometric measurement which only requires one optical filter for the measurement.
- light intensity for the donor or the acceptor is determined using the appropriate optical filter and another measurement of the samples is made without the use of any filter (intensity of the open spectrum). In this latter measurement, total light output (for all wavelengths) is quantified. Ratio calculations are then made using either equation 3 or 4. For the equation 3, only the optical filter for the acceptor is required. For the equation 4, only the optical filter for the donor is required.
- equations 1 through 4 can be derived from equations 1 through 4.
- one such derivative involves correcting for background light present at the emission wavelength for bioluminescent protein and/or acceptor molecule.
- the BRETCount instrument is a modified TopCount, wherein the TopCount is a microtiterplate scintillation and luminescence counter sold by Packard Instrument (Meriden, CT). Unlike classical counters which utilise two photomultiplier tubes (PMTs) in coincidence to eliminate background noise, TopCount employs single- PMT technology and time-resolved pulse counting for noise reduction to allow counting in standard opaque microtiterplates. The use of opaque microtiterplates can reduce optical crosstalk to negligible level. TopCount comes in various formats, including 1, 2, 6 and 12 detectors (PMTs) which allow simultaneous reading of 1, 2, 6 or 12 samples, respectively.
- PMTs photomultiplier tubes
- BRET Beside the BRETCount, other commercially available instrument are capable of performing BRET: the Victor 2 (Wallac, Finland (Perkin Elmer Life Sciences)) and the Fusion (Packard Instrument, Meriden).
- BRET can be performed using readers that can detect at least the acceptor molecule emission and preferably two wavelengths (for the acceptor molecule and the bioluminescent protein) or more.
- BRET is detected using a microfluidics device.
- Microfluidics devices conveniently require only an aliquot of the sample, generally not more than about 50 ⁇ L, to be transferred to the sample reservoir of the micro fluidics device. This is performed either manually or by pneumatic injection via a syringe, capillary or the like.
- the microfluidics device may contain a self-contained disposable biochip with patterned microchannels and compartments having storage means for storing a plurality of samples, reagents, and substrates. The steps of transferring sequentially at least one of the samples, or reagents, and then luminescent substrate from compartments through microchannels to the reaction sites could be automated. The luminescent substrates would then react with the donor molecules resulting in luminescence, which would be detected by an optical detector.
- An example of a microfluidics device for detecting luminescence is described in US 6,949,377.
- the present invention can be used to detect a wide variety of compounds, particularly odorants.
- the odorant will be a volatile organic or inorganic compound or inorganic gas that may be detected by chemosensory odorant receptors of at least one organism.
- chemosensory odorant receptors of at least one organism.
- These may include amine- and/or sulphydyrl-containing compounds, carboxylic acids, alcohols, aldehydes, alkanes, alkenes, aromatic compounds, esters, terpenes or terpene-derivatives, ethers, CO 2 etc. as well as compounds bearing combinations of these features.
- Odorants may be indicative of some biological or chemical state of value or of interest to humans. Such indications may include:
- the compound only binds the receptor portion of a polypeptide described herein.
- the polypeptide defined herein comprising an odorant receptor is used in a biosensor to detect a compound(s) of interest.
- a biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component. It typically consists of three parts, firstly at least one polypeptide defined herein comprising a G protein coupled receptor. Second, a transducer or detector element is required, which works in a physicochemical way (eg. optical, electrochemical) that transforms the signal resulting from the interaction of the compound with the polypeptide into another signal (i.e. transducers) that can be more easily measured and quantified. Third, an associated electronic or signal processor is required, which then displays the results of the interaction in a user-friendly way. An example of a biosensor involving BRET is described in Charest et al. (2005).
- the methods of screening are used for drug discovery and/or development.
- the receptor is a target for potential therapeutics.
- the receptor is a clinically important molecule such as, but not limited to, an adrenergic receptor, a serotinin receptor, a dopamine receptor, metabotropic/glumtamte receptor, a GABA receptor, a vomeronasal receptor, a taste receptor, or a secretin-like receptor.
- the present invention can also be mutliplexed.
- two or more polypeptides comprising different G protein coupled receptors are provided which bind different compounds.
- Each different G protein coupled receptor is labelled with a different bioluminescent protein and/or acceptor molecule such that they emit at different wavelengths to enable the detection and quantification of different target compounds.
- BRET receptor constructs were generated with BRET components inserted into the third intracellular loop (IC3) and at the C-terminus. Two BRET constructs were made with; (1) GFP in the middle of IC3 and RLuc(h) on the C-terminus; (2) OR
- IC3 The position of the third intracellular loop (IC3) was determined using TMAP on The Biology Workbench a web-based tool for prediction of transmembrane segments (http://seqtool.sdsc.edu).
- OGOR The amino acid sequence for this construct referred to as OGOR, is provided as SEQ ID NO: 15 (encoding nucleotide sequence provided as SEQ ID NO: 16).
- ODR-IO was amplified from C. elegans cDNA, which was prepared with standard techniques using superscript II (Invitrogen). PCR conditions were as follows 94°C 2mins, 30 cycles of 94°C 15secs, 59°C 30secs, 68°C 70sec, and a final extension step of 68°C for 5min. Standard Pfx50 PCR ingredients were used with primers ODR- lOXbalF 5 '-AGTCTAGAATGTCGGGAGAATTGTGGATTA-S ' (SEQ ID NO: 21) and ODR-10-attbl-R 5'-
- BRET constructs were constructed by PCR amplification of individual fragments of the construct (see Table 4 for primers and annealing temperatures).
- TM was performed with Pfx50 standard ingredients (Invitrogen) and conditions were as follows 94°C 2min, 30 cycles of 94°C 15sec, 59°C 30sec, 68°C 70sec, and a final extension step of 68°C for 5min. Separate components of the constructs were amplified using overlapping primers.
- Components were then purified and placed in a paired reaction to denature at 94°C, anneal 52°C (at overlapping primer sites) and extend 68 0 C for 5min for the required pairs. This was then used as template for PCR using the most 5' and 3' primers of the pairs. This was repeated until the full length constructs were made. They were then cloned into pGEM-T for sequencing. Error free clones were subcloned into pDONR201 (Invitrogen) using ApaLI. These were then subcloned into pYES- DEST52 (Invitrogen) using Gateway® technology (attB sites were included in original 5' and 3' primers) for subsequent expression in S. cerevisiae.
- PCR was performed with Pfx50TM standard ingredients (Invitrogen) and conditions were as follows 94°C 2mins, 16 cycles of 94°C 30secs, 55°C 1 minute, 68 0 C 6.8 minutes. Following sequencing the OGOR mutant was then subcloned into pYES- DEST52 (Invitrogen) using Gateway® technology (5' and 3' attB sites were included in original OGOR sequence) for subsequent expression in S. cerevisiae.
- YPD medium Add 2Og of bacteriological peptone, 1Og of yeast extract to 1 L of deionized water. Autoclave for 15 minutes.
- YPD plates Add 2Og of bacteriological peptone, 1Og of yeast extract and 20 g of agar to 1 L of deionized water. Autoclave for 15 minutes. Following autoclaving add 20 mL of 20 % glucose and pour agar plates.
- Yeast Synthetic Drop-Out Media without Uracil (SCMM-U) medium Add 6.7 g of yeast nitrogen base without amino acids (Product no. Y 0626, Sigma) and 1.92 g of yeast synthetic drop-out media supplement without uracil (Product no. Y 1501) to 1 L of deionized water. Autoclave for 15 minutes.
- SCMM-U plates Add 1.34 g of yeast nitrogen base without amino acids (Product no. Y 0626, Sigma), 0.384 g of yeast synthetic drop-out media supplement without uracil (Product no. Y 1501, Sigma) and 4 g of agar to 180 mL of deionized water. Autoclave for 15 minutes. Following autoclaving add 20 mL of 20% glucose and pour agar plates.
- Coelenterazine 400a Dissolve 1 mg of Coelenterazine 400a (Clz400a, Biosynth) in 10.20 mL of pure ethanol to make a 250 ⁇ M stock solution. Pipette 40 or 400 ⁇ L aliquots of Coelenterazine 400a into microcentrifuge tubes and then dry down with a
- Odorant Preparation - A 1 % stock solution of the odorants diacetyl, benzaldehyde and octanal were made up in the respective solvents water, ethanol and DMSO. The odorants were serially diluted in water to give the desired final concentration. The same dilutions of solvents without the addition of the odorants were also prepared.
- Yeast transformations were carried out using a yeast transformation kit (YEAST-I, Sigma).
- a YPD plate was streaked with INVSCl (Invitrogen) 5". cervesiae strain and incubated at 28 C for 2-3 days. Scrape a loop of InVSCl cells from the YPD plate into a microcentrifuge tube. Resuspend cells in 0.5 mL of transformation buffer (Product code T 0809) and spin for 5 seconds. Remove the supernatant, leaving 50 - 100 ⁇ L of the buffer in the tube. Add 10 ⁇ L of 10 mg/mL salmon testes DNA (Product code D 9156) to the tube.
- the OD of the overnight culture was determined. The amount of overnight culture required to obtain an OD 600 of 0.4 in 30 mL of induction medium (SCMM-U,
- D-PBS Dulbecco's phosphate buffered saline
- 1 x PBS containing 1000 mg/L glucose, 36 mg/L sodium pyruvate, calcium and magnesium (Invitrogen).
- the cells were lysed by French press ( ⁇ 18000 psi) and the soluble protein fractions were isolated by centrifugation at 9300 x g (4 C) for 15 minutes.
- soluble protein fraction was transferred to 13.15 mL polycarbonate thick wall centrifuge tubes and ultracentrifuged at 40,000 rpm (Beckman Coulter L-80 ultra-centrifuge) for 1 hour at 4 C. The supernatant was decanted and the membrane pellet resuspended in 1 mL of D-PBD or 1 x PBS, and left overnight at 4 C to solubilise.
- RET analysis was carried out in 96-well plates (Perkin-Elmer) and involved incubating the specified dual tagged receptor protein (OGOR) for 45 minutes with 1 ⁇ M of each particular odorant.
- the final volume was 100 ⁇ L and included 10 ⁇ L of odorant, receptor protein sample (see 'Normalization of sample concentration ' section), D-PBS (or 1 x PBS) and 5 ⁇ L of Clz400a (5 ⁇ M).
- a background signal was recorded by assaying a sample of D-PBS (or 1 x PBS).
- the 96-well plates Perkin-Elmer
- TM plate wells were sealed with Topseal - A (Packard) with each sample area individually sealed.
- Non odorants samples water, pyruvate, citric acid, lactic acid
- Non odorants solutions were prepared in the fume hood and assayed in the following order: 2-butanone, 2,3 - pentanedione, 2,3- butanediol and diacetyl.
- Clz400a substrate (5 ⁇ M) was added following the 45 minute incubation time and the signal recorded using a 0.50s integration time over a 5 second period. Following addition of Clz400a the well was re-sealed while the signal was recorded to reduce cross-contamination of samples. The assays were sequentially carried out in the order the samples were added to the wells.
- BRET ratio is calculated as emission ratio measured as ((emission at 515 nm for receptor sample)-(emission at 515 nm for D-PBD (or 1 x PBS))/(emission at 410 nm for receptor sample)- (emission at 410 nm for D-PBD) (or 1 x PBS)).
- the OGOR concentration was normalized to 10 nM by GFP 2 intensity.
- a dose response curve was constructed by varying odorant concentrations over the concentration range as indicated in the text. The lowest concentration was always assayed first and the highest last.
- a sigmoidal dose response curve was fitted, and EC50 calculated, by non-linear regression analysis using GraphPad Prism for Windows XP.
- the constructs produced have the BRET donor and acceptor inserted within the third intracellular loop and at the C-terminus of the odorant receptor ( Figure 1).
- Diacetyl binding by ODR-IO would cause an increase in the BRET ratio if the donor and acceptor components moved apart or an increase in the signal if the components moved closer together.
- the largest change in the BRET response to ligand binding of 37.5% is substantially greater than the observed change in the FRET signal ( ⁇ 5%) upon binding of 10 ⁇ M norepinephrine by the ⁇ 2A receptor (Lohse et al., 2003) and 1 ⁇ M PTH by
- the effect of changing odorant concentrations on the amplitude of the change in the BRET 2 signal of OGOR provides the first demonstration of a dose- dependent response.
- the EC50 value was calculated to be 3.55 fM diacetyl, which is equivalent to 0.31 parts per quadrillion (ppq).
- the cell free assay presented here is more than nine orders of magnitude more sensitive for diacetyl quantification compared to whole cell assays and existing chemical detection systems such as fluorescence detection (Li et al., 2009) and a gas chromatographic technique with flame ionization detection (GC-FID) (Macciola et al., 2008).
- fluorescence detection Li et al., 2009
- GC-FID gas chromatographic technique with flame ionization detection
- the BRET components used to tag the ODR-IO receptor were replaced with FRET components resulting in the insertion of CFP into the 3 rd intracellular loop and YFP at the C-terminus (OCOY).
- the CFP (mCFP) (SEQ ID NO:45) and YFP (mCitrine) (SEQ ID NO:46) derivatives were both monomeric and codon optimized (EUROSCARF) for yeast expression (SEQ ID NOs 47 and 48 respectively).
- the OCOY sequence flanked by Kpnl and Xhol restriction sites was synthesised by Genscript.
- the OCOY sequence was inserted into the Kpnl and Xhol sites of the pENTRl 1 vector. These were then recombined into pYES-DEST52 (Invitrogen) using Gateway ® technology for subsequent expression in S. cerevisiae.
- the OCOY coding sequence is provided as SEQ ID NO:49, whereas the amino acid sequence is provided as SEQ ID NO: 5 O.
- the fluorescence intensity of the sample was determined from spectral scans and the sample amount normalized by the OCOY fluorescence intensity.
- the YFP intensity (530 nm) was normalized to 721.32 a.u. for OCOY. 50 ⁇ L of OCOY was pipetted into the appropriate well and the volume made up to 100 ⁇ L with 1 x PBS.
- the F ⁇ rster distance (Ro), the RET probe separation corresponding to 50% of the maximum RET efficiency, for the BRET 2 system was recently determined to be 7.5 nm, the largest Ro value determined for any genetically encoded RET pair (Dacres et al., 2009).
- the Ro value for the FRET system was previously determined to be 4.8 nm (Evers et al., 2006) indicating that the BRET 2 system is able to probe a larger distance range (3.8 - 11.3 nm) than the FRET system (2.4 - 7.2 nm).
- the measured transfer efficiency for OGOR changed from 64.3% to 47% upon the addition of diacetyl (1 ⁇ M) indicating a distance change of 6.8 nm to 7.6 nm.
- the TGTR sequence flanked by Ncol and Notl restriction sites was synthesised by Genscript.
- the TGTR sequence was inserted into the Ncol and Notl sites of the pE ⁇ TRl 1 vector. These were then recombined into pYES-DEST52 (Invitrogen) using Gateway ® technology for subsequent expression in S. cerevisiae.
- TGTR in InVSCl
- OGOR expression The same expression protocol was used for the expression of TGTR in InVSCl as for OGOR expression.
- the TGTR coding sequence is provided as SEQ ID ⁇ O:51, whereas the amino acid sequence is provided as SEQ ID NO: 52.
- the bacterial strain OP50 was grown in LB overnight at 37°C with shaking (200 rpm). E.coli strain OP50 is usually used as a food source for nematode growth in the laboratory (Brenner, 1974). Two assays were performed using the OP50 bacterial culture. The first assay was the addition of 10 ⁇ L of the bacterial culture or 10 ⁇ L of LB to 10 nM TGTR. The second assay required that 1 mL of the bacterial culture was placed in a microcentrifuge tube and centrifuged at 10000 x g for 1 minute.
- ODR-10 transduction cassette is engineered so the ODR-10 N-terminus (aal-32) and IC3 to C terminus (aa225 -aa339) with BRET tags flank a multiple cloning site ( Figure 11).
- the chimeric receptors are expressed in yeast. Ligand binding regions of ORs are shuffled "in-frame' into this cassette and these chimeric receptors can be used to assess ligand binding by BRET.
- OdrlO labelled with Rluc at the C-terminus SEQ ID NO: 17
- OdrlO labelled with GFP2 at the C-terminus SEQ ID NO: 19
- a colony of transformed yeast cells was grown in yeast synthetic drop-out medium (SCMM, Sigma) lacking appropriate nutrients (such as uracil (U), tryptophan (T)) to select for plasmid-containing cells with 2% glucose for overnight at 28°C with shaking 190 rpm; SCMM-U-T for Invscl co-expressed />YesDest52-OdrlO-Rluc and j pDestpESC-Odrl0-GFP2 (Invscl /OR/OG), SCMM-U for Invscl expresses p ⁇ 2U- odrlO-Rluc (Invscl/OR) alone and />YesDest52 odrlO-GFP2 (Invscl/OG) alone respectively.
- SCMM yeast synthetic drop-out medium
- appropriate nutrients such as uracil (U), tryptophan (T)
- OD600 was determined and the cells were suspended in induction medium (with 2% galactose and 1% raffinose instead of glucose as the culture medium) to give a final OD600 of 0.4.
- This induction culture was incubated for 24 hours with shaking at 15°C to induce fusion receptor expression.
- the culture was then pelleted and washed twice with cold phosphate-buffered saline (PBS). Cell pellet can be stored at -80 0 C and suspended in PBS buffer just before the BRET 2 assay.
- First Invscl/OR/OG was quantified by GFP intensity (about 3000 RLU or c.p.s in 100 ⁇ l per well) by direct excitation of GFP at Ex420nm in a white 96-well microplate (Perkin Elmer) using a SpectraMax M2 spectrofluorometer (Molecular Devices).
- the Renilla luciferase activity of the quantified Invscl/OR/OG cells was determined by adding luciferin coelenterazine native (final concentration 5 ⁇ M) into a total volume of 100 ⁇ l cell in a 96-well white microplate and measuring luminescence signals immediately (Polarstar Optima, BMGLABTECH).
- Invscl/OG was normalised to have the similar GFP intensity as Invscl/OR/OG.
- Invscl/OR was normalised to have the similar Rluc activity as Invscl/OR/OG.
- Negative control host strain
- Invscl was quantified to have same cell density as Invscl/OR/OG.
- Assay procedure was modified from Issad and Jockers (2006) and conducted in a 96-well white microplate in a total volume of 100 ⁇ l and two repeat for each sample. Three biological independent assays will be conducted and data are pooled.
- Coelenterazine h DeepBlueC substrate Biosynth AG was added at a final concentration of 10 ⁇ M, and readings were performed with a dual wavelength lumino/fluorometer mircoplate reader (BMGLabtech). The following optimized filter settings were used to measure Rluc light emission (410+80 nm) and GFP 2 light emission (515+30 nm).
- the BRET ratio was defined as the difference of the emission at 515nm/410nm of co-expressed Rluc and GFP2 fusion proteins and the emission at the 515nm/410nm of the Rluc fusion protein alone. Results were expressed in milliBRET units, 1 milHBRET corresponding to the BRET ratio value multiplied by 1000 (Ayoub, 2002). Illumination of control cells (host strain Invscl) that do not express a Rluc or GFP2 fusion at the same settings results in detection of cell autofluorescencee. Subtraction of the autofluorescence from all tested samples was taken place before BRET ratio values were calculated.
- Properly inducted yeast cells (GFP 2 level was not more than 2 fold over background of host strain Invscl cells) were washed three times with ice-cold Buffer A (75 mM tris-HCl, pH7.4, 12 mM MgC12 and 2 mM EDTA) and the final pellet was suspended in Buffer B (Buffer A + protease inhibitor cocktail, complete Mini EDTA- free (Roche Applied Science)). Then the yeast cells are disrupted using a French Press and centrifuged for 15 mins at 10,000 x g at 4°C. The supernatant was ultracentrifuged for 1 hour at 130000 x g and the pellet was suspended in cold Buffer B.
- Buffer A 75 mM tris-HCl, pH7.4, 12 mM MgC12 and 2 mM EDTA
- Buffer B Buffer A + protease inhibitor cocktail, complete Mini EDTA- free (Roche Applied Science)
- Membrane protein solubilisation and immunoprecipitation procedures were modified according to the method described by Ayoub et al. (2002). Crude membrane was solubilised with 0.5% digitonin (Sigma) and this mixture was stirred for 3h at 4°C. Non-solubilised membrane proteins were removed by centrifugation at 18,000 x g for 30 mins at 4°C. The digitonin concentration was adjusted to 0.2% using cold Buffer B and the polyclonal anti-GFP antibodies (Sapphire Bioscience, Cat No 120-29000) were added to a final dilution of 1: 1000.
- the mouse 0C 2A adrenergic receptor (SEQ ID NO: 56) was engineered so that the GFP 2 was inserted into the 3 rd intracellular loop with its N-terminus following Ala250 and its C-terminus adjacent to Arg372. RLuc was fused to Val461 in the C-terminus of the receptor (amino acid sequence provided as SEQ ID NO:54 and encoding open reading frame as SEQ ID NO:53).
- the dual-tagged adrenergic receptors are expressed in yeast. These receptors can be used to monitor ligand binding by the mouse 0C 2A adrenergic receptor via changes in the BRET 2 ratio.
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JP2012516429A (en) | 2012-07-19 |
US8647887B2 (en) | 2014-02-11 |
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EP2382470A1 (en) | 2011-11-02 |
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HK1163254A1 (en) | 2012-09-07 |
US20120077210A1 (en) | 2012-03-29 |
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