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WO2001068706A1 - Proteines chimeres et de fusion du recepteur de l'hormone de concentration de la melanine - Google Patents

Proteines chimeres et de fusion du recepteur de l'hormone de concentration de la melanine Download PDF

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WO2001068706A1
WO2001068706A1 PCT/US2001/008071 US0108071W WO0168706A1 WO 2001068706 A1 WO2001068706 A1 WO 2001068706A1 US 0108071 W US0108071 W US 0108071W WO 0168706 A1 WO0168706 A1 WO 0168706A1
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seq
protein
mch
nucleic acid
concentrating hormone
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Donald J. Marsh
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Merck and Co Inc
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Merck and Co Inc
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Priority to JP2001567796A priority Critical patent/JP2003532388A/ja
Priority to EP01922374A priority patent/EP1265926A1/fr
Priority to CA002402147A priority patent/CA2402147A1/fr
Priority to US10/221,461 priority patent/US7029878B2/en
Publication of WO2001068706A1 publication Critical patent/WO2001068706A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • MCH Melanin- concentrating hormone
  • MCH has been localized primarily to neuronal cell bodies of the hypothalamus which are implicated in the control of food intake, including perikarya of the lateral hypothalamus and zona inertia. (Knigge et al, 1996. Peptides 17, 1063- 1073.)
  • MCH mRNA is up regulated in fasted mice and rats and in the ob/ob mouse.
  • ICN MCH centrally
  • MCH antagonizes the hypophagic effects seen with -melanocyte stimulating hormone (odVISH).
  • odVISH -melanocyte stimulating hormone
  • MCH action is not limited to modulation of food intake as effects on the hypothalamic-pituitary-axis have been reported. ( ⁇ ahon 1994. Critical Rev. in Neurobiol 8, 221-262.) MCH may be involved in the body response to stress as MCH can modulate the stress-induced release of CRF from the hypothalamus and ACTH from the pituitary. In addition, MCH neuronal systems may be involved in reproductive or maternal function.
  • the present invention features melanin concentrating hormone receptor
  • MCH-R chimeric and fusion proteins.
  • MCH-R chimeric proteins comprise an
  • MCH-R polypeptide region made up of at least two or more polypeptide regions characteristic of MCH-R found in different species.
  • MCH-R fusion proteins comprise an MCH-R polypeptide region and a fluorescent protein region.
  • An MCH-R polypeptide region provides a functional G-protein coupled receptor region able to bind MCH and transduce an intracellular signal.
  • MCH-R polypeptide regions include naturally occurring MCH-R, chimeric MCH-R containing two or more regions from naturally occurring MCH-R, and functional derivatives thereof.
  • a first aspect of the present invention describes a fusion protein comprising (a) an MCH-R polypeptide region and (b) a fluorescent polypeptide region.
  • the fluorescent polypeptide region is joined directly, or though a polypeptide linker, to the carboxy side of the MCH-R polypeptide region.
  • the protein comprises: (a) an MCH-R binding region characteristic of a human MCH-R, (b) a transmembrane domain characteristic of a non-human MCH-R, and (c) an intracellular domain characteristic of a non-human MCH-R.
  • nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein.
  • Such nucleic acid comprises either a contiguous nucleotide sequence that codes for the protein or a sequence that is processed by a host cell to produce a contiguous nucleotide sequence encoding for the protein. Processing of a nucleic acid sequence to produce a contiguous nucleotide sequence encoding for a protein can occur by the splicing together of exons resulting in intron removal.
  • Another aspect of the present invention describes an expression vector comprising a nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein.
  • Another aspect of the present invention describes a recombinant cell comprising nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein.
  • the nucleic acid may be part of the host genome or may exist independently of the host genome.
  • Another aspect of the present invention describes a non-human transgenic animal comprising nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein.
  • Another aspect of the present invention describes a method for assaying for MCH-R active compounds by measuring the effect of a test preparation on one or more MCH-R activities. The method is performed using either an MCH-R fusion protein or an MCH-R chimeric protein described herein.
  • Figure 1 illustrates aequorin assay results comparing a mouse MCH-R fusion with a human wild type MCH-R and a CMV-EGFP control.
  • Figure 2 illustrates a cAMP flashplate assay of CHO cell clones stably expressing mMCH-lR-EGFP.
  • Cells from individual clones were dissociated in enzyme free media and stimulated for 15 minutes at 37°C with human MCH at the indicated concentrations in the presence of 10 ⁇ M forskolin. Cells were then lysed and assayed for bound [ 125 I]cAMP.
  • Mouse MCH-IR-EGFP clones exhibited EC50 values (0.1111, 0.1255, 0.1291, or 0.2304 nM) indistinguishable from that of a CHO cell clone expressing the wild-type human short isoform of MCH-IR ( 0.1282 nM).
  • Figure 3 illustrates a cAMP flashplate assay of CHO cell clones stably expressing human short/mouse species chimeric MCH-IR-EGFP.
  • Cells from individual clones were dissociated in enzyme free media and stimulated for 15 minutes at 37°C with human MCH at the indicated concentrations in the presence of 10 ⁇ M forskolin. Cells were then lysed and assayed for bound [ 125 I]cAMP.
  • the present invention features MCH-R chimeric and fusion proteins. Such proteins have a variety of different uses including being used as a research tool to study MCH-R function and dynamics, and being used to screen for MCH-R agonists and antagonists.
  • the MCH-R provides a target to achieve different beneficial effects in a patient.
  • MCH-R activity is modulated to achieve one or more of the following: weight loss, weight gain, treat cancer (e.g., colon or breast), reduce pain, treat diabetes, reduce stress, or teat sexual dysfunction.
  • Modulation of MCH-R activity can be achieved by evoking a response at the MCH receptor or by altering a response evoked by an MCH receptor agonist or antagonist.
  • Compounds modulating MCH-R receptor activity include agonists, antagonists, and allosteric modulators.
  • MCH-R antagonists and allosteric modulators negatively affecting activity will be used to achieve weight loss, treat cancer (e.g., colon or breast), reduce pain, reduce stress, or teat sexual dysfunction; and MCH-R agonists and allosteric modulators positively affecting activity will be used to produce a weight gain.
  • MCH-R activity is modulated to achieve a weight loss or to treat diabetes in a patient.
  • Diabetes mellitus can be treated by modulating MCH-R activity to achieve, for example, one or both of the following: enhancing glucose tolerance or decreasing insulin resistance.
  • Excessive body weight is a contributing factor to different diseases, including hypertension, diabetes, dyslipidemias, cardiovascular disease, gall stones, osteoarthritis, and certain forms of cancers. Bringing about a weight loss can be used, for example, to reduce the likelihood of such diseases and as part of a treatment for such diseases. Weight reduction can be achieved by modulating MCH-R activity to obtain, for example, one or more of the following effects: reducing appetite, increasing metabolic rate, reducing fat intake, or reducing carbohydrate craving.
  • Increasing body weight is particularly useful for a patient having a disease or disorder, or under going a treatment, accompanied by weight loss.
  • diseases or disorders accompanied by weight loss include anorexia, AIDS, wasting, cachexia, and frail elderly.
  • treatments accompanied by weight loss include chemotherapy and radiation therapy.
  • MCH-R chimeric Proteins contain an MCH-R polypeptide region made up by at least two or more polypeptide regions characteristic of MCH-R found in different species.
  • the different polypeptide regions that are present provide for an N ⁇ terminal extracellular domain; a transmembrane domain made up of transmembrane regions, extracellular loop regions, and intracellular loop regions; and an intracellular carboxy terminus domain.
  • MCH-R amino acid sequences include the following: SEQ. ID. NO. 1 (human MCH1R long form), SEQ. ID. NO. 2 (human MCH1R short form), and SEQ. ID. NO. 3 (mouse MCH1R).
  • the MCH-R chimeric protein comprises an MCH-R binding region characteristic of a human MCH-R along with transmembrane and intracellular domains characteristic of a non-human MCH-R.
  • MCH-R binding region characteristic of a human MCH-R along with transmembrane and intracellular domains characteristic of a non-human MCH-R.
  • the presence of a human MCH-R binding region provides for a "humanized" MCH-R chimeric receptor.
  • transmembrane and intracellular domains characteristic of a non-human MCH-R can be used in conjunction with a non-human host to provide a more naturally occurring environment for these regions.
  • an MCH-R chimeric having mouse transmembrane and intracellular domains are preferably used in murine cells lines or in transgenic mice.
  • MCH-R chimeric proteins may contain regions other than extracellular, transmembrane, and intracellular domains that do not substantially decrease the activity of the protein.
  • additional regions do not cause a decrease of more than about 25% of MCH-R activity as measured using one or more of the assays described in the examples provided below.
  • additional regions include fluorescent protein regions and linker regions.
  • the MCH-R chimeric protein comprises: (a) an MCH binding region characteristic of a first species and (b) a transmembrane and intracellular domain region characteristic of a second species joined directly, or though a linker, to the carboxy side of the MCH binding region.
  • the protein comprises, consists, or consists essentially of an MCH-R polypeptide having a sequence similarity of at least about 75%, at least 85%, or at least 95% with either SEQ. ID. NO. 4 (human short form/mouse species chimeric MCHIR) or SEQ. ID. NO. 5 (human long form/mouse species chimeric).
  • the protein comprises, consists essentially of, or consists of, SEQ. ID. NO. 4 or SEQ. ID. NO. 5.
  • Sequence similarity for polypeptides can be determined by BLAST. (Altschul et al, 1997. Nucleic Acids Res. 25, 3389-3402, hereby incorporated by reference herein.) In an embodiment of the present invention, sequence similarity is determined using tBLASTn search program with the following parameters: MATRIX:BLOSUM62, PER RESIDUE GAP COST: 11, and Lambda ratio: 1.
  • Amino acids can be divided into different groups as follows: neutral and hydrophobic (alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine); neutral and polar (glycine, serine, threonine, tyrosine, cysteine, asparagine, and glutamine); basic (lysine, arginine, and histidine); and acidic (aspartic acid and glutamic acid). Generally, in substituting different amino acids it is preferable to exchange amino acids having similar properties.
  • such changes are made taking into account the position of the amino acid to be substituted in the polypeptide.
  • arginine can substitute more freely for nonpolor amino acids in the interior of a polypeptide then glutamate because of its long aliphatic side chain.
  • MCH-R fusion proteins contain an MCH-R polypeptide region and a fluorescent protein region either directly joined together or joined together through a linker. These regions provide MCH-R activity and a marker for evaluating MCH-R dynamics.
  • An MCH-R polypeptide region provides functional MCH-R activity and includes naturally occurring MCH-R, chimeric MCH-R, and derivatives thereof.
  • Preferred derivatives thereof have a sequence similarity of at least about 75%, at least about 85%, or at least about 95% to a naturally occurring MCH-R or a chimeric
  • a fluorescent protein region contains a chromophore that fluoresces.
  • the fluorescent protein region is the green fluorescent protein of the jellyfish Aequorea victoria or a derivative thereof.
  • Preferred derivatives have a sequence similarity of at least about 75%, at least about 85%, or at least about 95% to the Aequorea victoria green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • the Aequorea victoria green fluorescent protein and examples of derivatives thereof are described by Cormack et al, 1996. Gene 17, 33-38; Yang et al, 1996. Nucleic Acids Research 24, 4592-4593;
  • the MCH-R polypeptide region comprises, consists essentially of, or consists of, a sequence selected from the group consisting of: SEQ. LD. NO. 1, SEQ. D. NO. 2, SEQ. LD. NO. 3, SEQ. LD. NO. 4, and SEQ. ID. NO. 5; and the fluorescent polypeptide region comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of SEQ. ID.
  • the optionally present linker is a polypeptide region that is preferably from 1 to about 100 amino acids in length. In different embodiments the linker is up to 75, 50 or 25 amino acids in length.
  • the MCH-R fusion protein comprises, consists essentially of, or consists of, the MCH-R polypeptide region and the fluorescent polypeptide region. More preferably, the protein comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of: SEQ. ID. NO. 11 (mouse MCHlR-linker-EGFP), SEQ. ID. NO. 12 (mouse MCHIR/EGFP direct fusion), SEQ. D. NO. 13 (human short form/mouse species chimeric MCHlR-linker- EGFP), or SEQ. ID. NO. 14 (human long form/mouse species chimeric MCH1R- linker-EGFP).
  • MCH-R chimeric and fusion proteins can be produced using techniques well known in the art. Preferably, such proteins are produced by recombinant expression inside a host cell by way of an expression vector or by way of nucleic acid integrated into the host genome. Examples of nucleic acid sequences encoding for MCH-R polypeptide regions, fluorescent protein regions, MCH-R chimeric proteins, and MCH-R fusion proteins are provided for by SEQ. D. NOs. 15-
  • An expression vector contains recombinant nucleic acid encoding for a polypeptide along with regulatory elements for proper transcription and processing.
  • the recombinant nucleic acid contains two or more nucleic acid regions not naturally associated with each other. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing recombinant nucleic acid in a particular host.
  • Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids, and viruses.
  • an expression vector includes a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. Another preferred element is a polyadenylation signal providing for processing in eukaryotic cells.
  • an expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number.
  • Mammalian expression vectors including pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), pCI- neo (Promega) and .lambda.ZD35 (ATCC 37565).
  • Bacterial expression vectors well known in the art include pETlla (Novagen), lambda gtll (Invitrogen), pcDNAJJ (Invitrogen), and pKK223-3 (Pharmacia).
  • Fungal cell expression vectors well known in the art include pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
  • Insect cell expression vectors well known in the art include Blue Bac HI (Invitrogen).
  • Recombinant host cells may be prokaryotic or eukaryotic.
  • recombinant host cells include the following: bacteria such as E. coli; fungal cells such as yeast; mammalian cells such as human, bovine, porcine, monkey, hampster, and rodent; and insect cells such as Drosophila and silkworm derived cell lines.
  • L cells L-M(TK.sup.-) ATCC CCL 1.3
  • L cells L-M ATCC CCL 1.2
  • 293 ATCC CRL 1573
  • Raji ATCC CCL 86
  • CV-1 ATCC CCL 70
  • COS-1 ATCC CRL 1650
  • COS-7 ATCC CRL 1651
  • CHO-K1 ATCC CCL 61
  • 3T3 ATCC CCL 92
  • NIH/3T3 ATCC CRL 1658
  • HeLa ATCC CCL 2
  • C127I ATCC CRL 1616
  • BS-C-1 ATCC CCL 26
  • MRC-5 ATCC CCL 171
  • Expression vectors may be introduced into host cells using standard techniques. Examples of such techniques include transformation, transfection, lipofection, protoplast fusion, and electroporation. Nucleic acid encoding for a polypeptide can be expressed in a cell without the use of an expression vector employing, for example, synthetic mRNA or native mRNA. Additionally, mRNA can be translated in various cell-free systems such as wheat germ extracts and reticulocyte extracts, as well as in cell based systems, such as frog oocytes. Introduction of mRNA into cell based systems can be achieved, for example, by microinjection.
  • MCH-R is G-protein coupled receptor. Techniques for measuring different G-protein activities, such as Gi/o, Gs, and Gq are well known in the art. MCH-R activity is preferably assayed for by measuring either Gi/o or Gq. Gi/o and Gs activity can be measured using techniques such as a melonaphore assay, measuring cAMP production, measuring inhibition of cAMP accumulation, and measuring binding of 35S-GTP. cAMP can be measured using different techniques such as radioimmunoassay and indirectly by cAMP responsive gene reporter proteins.
  • Gq activity can be measured using techniques such as those measuring intracellular Ca .
  • techniques well known in the art that can be employed to measure Ca include the use of dyes such as Fura-2 and the use of Ca - bioluminescent sensitive reporter proteins such as aequorin.
  • An example of a cell line employing aequorin to measure G-protein activity is HEK293/aeql7. (Button et al, 1993. Cell Calcium 14, 663-671, and Feighner et al, 1999. Science 284, 2184- 2188, both of which are hereby incorporated by reference herein.)
  • Functional assays can be performed using individual compounds or preparations containing different compounds.
  • a preparation containing different compounds where one or more compounds affect MCH-R chimeric or fusion protein activity can be divided into smaller groups of compounds to identify the compound(s) affecting MCH-R chimeric or fusion protein activity.
  • a test preparation containing at least 10 compounds is used in a functional assay.
  • Functional assays can be performed using recombinantly produced
  • MCH-R chimeric or fusion protein present in different environments.
  • environments include, for example, cell extracts and purified cell extracts containing the MCH-R chimeric or fusion protein expressed from recombinant nucleic acid and an appropriate membrane for the polypeptide; and the use of a purified MCH-R chimeric or fusion protein produced by recombinant means that is introduced into a different environment suitable for measuring G-protein activity.
  • Fluorescent protein joined to an MCH receptor can be employed to study different aspects of receptor dynamics including receptor sequestration, receptor densitization, and receptor localization.
  • the fluorescent protein can be used in in vitro or in vivo systems.
  • SEQ. ID. NOs. 1-29 include examples of polypeptide and encoding nucleic acid sequences for MCH-R polypeptide regions, fluorescent polypeptide regions, fusion proteins and chimeric proteins. Some cases the encoding nucleic acid is shown with additional nucleic acid upsteam or downstream from an open reading frame.
  • SEQ. ID. NO. 1 Human long form MCHIR
  • SEQ. ID. NO. 2 Human short form MCHIR MDLEASLLPTGPNASNTSDGPDNLTSAGSPPRTGSISY ⁇ N ⁇ MPSVFGTICLLG ⁇ G
  • SEQ. ID. NO. 4 Human short form/mouse species chimeric MCHIR
  • SEQ. ID. NO.5 Human long form/mouse species chimeric MCHIR
  • SEQ. ID. NO. 7 EGFP
  • SEQ. ID. NO. 11 Mouse MCHlR-linker-EGFP MDLQASLLSTGPNASMSDGQDNFTLAGPPPRTRSVSYIN ⁇ MPSVFGTICLLGI VGNSTVJTAVVKKSKLHWCSNWD LNLSVVDLLFLLGMPFNIIHQLMGNGV WHFGETMCTLITAMDANSQF ⁇ STYILTAMAJ ⁇ RYLATVHPISSTKFRKPSMAT LVICLLWALSMSITPVWLYAI ⁇ IPFPGGAVGCG]TLPNPDTDLYWFTLYQFFLA FALPFVVTT'AAYVKILQPJVITSSVAPASQRSII ⁇ RTK-RVTRTAIAICLVFFVCWA PYYVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYTVLCETFRKRLVLS VKPAAQGQLRTVSNAQTADEERTESKGTVDGTAGPGSINTMVSKGEELFTGV VPILVELDGDV ⁇ GHK ⁇ SVSGEGEGDATYGKLT
  • SEQ. ID. NO. 12 Mouse MCHIR/EGFP direct fusion
  • SEQ. ID. NO. 13 Human short form/mouse species chimeric MCHlR-linker- EGFP
  • SEQ. ID. NO. 15 Human long form MCHIR cDNA
  • SEQ. ID. NO. 16 Human short form MCHIR cDNA
  • SEQ. ID. NO. 18 Mouse MCHIR genomic DNA
  • SEQ. ID. NO. 19 Human short form/mouse species chimeric MCHIR
  • SEQ. ID. NO. 21 Aequorea victoria Green Fluorescent Protein (GFP) cDNA
  • Nucleic acid sequence start and stop codons are highlighted and a 12 amino acid linker sequence is denoted in lower case: gtcgacggtaccgcgggcccgggatccatcgccaccATGGTGAGCAAGGGCGAGGAGCTGTT CACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC CCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACG
  • SEQ. ID. NO. 27 Mouse MCH1R EGFP direct fusion
  • SEQ. ID. NO. 28 Human short form mouse species chimeric MCHlR-linker- EGFP
  • SEQ. ID. NO. 29 Human long form/mouse species chimeric MCHlR-linker-
  • DNA vectors encoding fusion proteins between a MCH-R receptor (MCHIR) and several different superbright variants of Green Fluorescent Protein (GFP) were generated.
  • GFP variants were fused either via a 12 amino acid linker: TCGACGGTACCGCGGGCCCGGGATCCATCGCCACC (SEQ. ID. NO. 30), amino acid sequence: VDGTAGPGSIAT (SEQ. ID. NO. 31) (linker fusions) or directly to the C-terminus of MCHIR (direct fusions).
  • Mouse MCHlR-linker-GFP Variant Fusion Constructs Initially, mouse MCHIR was fused in frame via the linker to Enhanced Green Fluorescent Protein (EGFP). MCHIR was PCR-amplified (95°C for 5 minutes; 95°C for 30 seconds, 60°C for 45 seconds, 68°C for 3.5 minutes, for 15 cycles; 68°C for 7 minutes) from a full-length mouse MCHIR genomic DNA lambda clone utilizing a high fidelity polymerase mix (Expand High Fidelity PCR System from Boehringer Mannheim) and PCR primers [MCHIR (Eco RI) 5': GCGAATTCACCATGGATCTGCAAGCCTCG (SEQ. ID. NO.
  • the MCHIR N-terminal PCR primer was also designed to introduce a Kozak consensus sequence for translation which contained an Nco I site (5'-ACCATGG-3'), and the MCHIR C-terminal PCR primer was also designed to eliminate the endogenous stop codon present in the mouse MCHIR gene.
  • the resulting PCR product was phenol/chloroform extracted, restriction digested with Eco RI and Sal I, gel purified, and subcloned in frame into the multicloning site of Clontech's pEGFP- N3 vector between Eco RI and Sal I sites.
  • Several resulting clones for this construct were sequenced to identify a clone with an entirely correct nucleotide sequence. This clone was named mMCHlR-1-EGFP for mouse MCHlR-linker-EGFP.
  • pRSET-Emerald, pRSET-Topaz, and pRSET-WlB were obtained from Aurora Biosciences Co.
  • Sal I to Not I fragments containing either Emerald, Topaz, or W1B were excised from the resulting pBluescript-Emerald, pBluescript-Topaz, and pBluescript-WlB vectors, respectively.
  • Appropriate fragments were gel purified and subcloned into mMCHlR- 1-EGFP digested with Sal I and Not I, replacing the Sal I to Not I EGFP fragment with the corresponding Sal I to Not I fragment from either Emerald, Topaz, or W1B.
  • Several clones for each construct were sequenced to confirm the presence of the appropriate GFP variant.
  • the resulting vectors were named mMCHlR-1-Emerald, mMCHlR-1-Topaz, and mMCHlR-1-WlB for mouse MCHlR-linker-Emerald, mouse MCHlR-linker-Topaz, and mouse MCHlR-linker-WlB, respectively.
  • Mouse MCHIR/GFP Variant Direct Fusion Constructs A two step PCR strategy was employed to generate the direct fusion constructs. First, mouse MCHIR, EGFP, and Emerald were PCR-amplified from a full-length mouse MCHIR genomic DNA lambda clone, Clontech's pEGFP-N3 vector, and Aurora's pRSET-Emerald vector, respectively. Mouse MCHIR was PCR-amplified according to the previously mentioned conditions utilizing the same N-terminal PCR primer [MCHIR (Eco RI) 5': GCGAATTCACCATGGATCTGCA AGCCTCG (SEQ. ID. NO. 32)], but in this case a different C-terminal PCR primer was employed.
  • the C-terminal PCR primer [MCHIR (EGFP/Emerald) 3': CCTTGCTCACCATGGTGCCTTTGCTTTCTGTCC (SEQ. LD. NO. 34)] eliminated the endogenous stop codon of mouse MCHIR as before and introduced a region of nucleotide sequence complementary to the nucleotide sequence of the N-terminus of EGFP.
  • EGFP and Emerald were PCR-amplified (95°C for 5 minutes; 95°C for 30 seconds, 60°C for 45 seconds, 68°C for 1.5 minutes, for 15 cycles; 68°C for 7 minutes) separately with a high fidelity polymerase mix (Advantage HF-2 from Clontech) from their respective templates utilizing a common N-terminal PCR primer [EGFP/Emerald (MCHIR) 5': CAGAAAGCAAAGGCACCATGGTGAGCAA GGGCGAGGAGC (SEQ. ID. NO.
  • PCR reactions were set up between the previously generated mouse MCHIR and EGFP, or mouse MCHIR and Emerald PCR products. Following an initial 5 minute denaturation step at 95°C, two rounds of thermocycling (95°C for 30 seconds, 60°C for 45 seconds, 68°C for 4 minutes) were performed in the absence of PCR primers. This allowed the mouse MCHIR and GFP variants to anneal at their complementary regions and to be filled in by the high fidelity polymerase mix (Expand High Fidelity PCR System from Boehringer Mannheim), yielding double stranded template DNA.
  • the high fidelity polymerase mix Exand High Fidelity PCR System from Boehringer Mannheim
  • mice MCHIR mouse MCHIR (Eco RI) 5': GCGAATTCACCATGGATCTGCAAGCCTCG (SEQ. ID. NO. 32)
  • appropriate C-terminal PCR primers [EGFP 3': GGCGGATCCTCTAGAGTC GCGGCC (SEQ. ID. NO. 36) or Emerald (EGFP) 3': GCTCTAGAGTCGCGG CCGCTTACTTGTACAGCTCGTCC (SEQ. LD. NO. 37)] were added to the reactions and thermocycling was continued for an additional fifteen cycles followed by a final extension at 68°C for 7 minutes.
  • the resulting PCR products were phenol/chloroform extracted, restriction digested with Eco RI and Not I, electrophoresed on an agarose gel, and appropriate fragments were gel purified.
  • Eco RI to Not I fragments represent direct fusions between either mouse MCHIR and EGFP, or mouse MCHIR and Emerald.
  • Clontech's pEGFP-N3 vector was restriction digested with Eco RI and Not I liberating an approximately 780 bp Eco RI to Not I EGFP fragment. This restriction digest was electrophoresed on an agarose gel and the approximately 3.9 Kb pEGFP-N3 vector backbone was gel purified.
  • MCHIR/Emerald direct fusion fragments were subcloned into the pEGFP-N3 vector backbone between Eco RI and Not I sites.
  • Several resulting clones for each of these two constructs were sequenced to identify clones with correct nucleotide sequence; however, no clones with entirely correct nucleotide sequences were identified.
  • Several clones for each of the two constructs only had nucleotide mismatches in the intron region of mouse MCHIR, and therefore, were not expected to effect the functionality of the resulting fusion proteins.
  • These clones were named mMCHlR/EGFP and mMCHlR/Emerald for mouse MCHIR/EGFP direct fusion and mouse MCHIR/Emerald direct fusion, respectively.
  • MCHlR-linker-GFP Variant Fusion Constructs The initial mouse MCHlR-linker-GFP variant fusion constructs were modified to generate both human short form and human long form/mouse species chimeric MCHlR-linker-GFP variant fusion constructs.
  • HEK293 Aequorin National Institutes of Health
  • CHO mammalian cell lines were transiently transfected with the various MCHIR/GFP variant fusion constructs, as well as the appropriate control constructs. Transfection was performed using Lipofectamine 2000 (Gibco BRL) per the manufacturer recommended protocol. Approximately 48 hours after transfection cells were harvested, stimulated with various concentrations of human MCH, and assayed for either aequorin bioluminescence (HEK293 Aequorin cells) or cAMP production (CHO cells). Aequorin bioluminescence is a representative measure of intracellular Ca 2+ mobilization. cAMP production was measured with the Adenylyl Cyclase Activation FlashPlate Assay (NEN Life Science Products, Inc.).
  • the resulting fusion protein exhibited functional activity comparable to that of the wild-type human MCHIR short form (MCH-R wt).
  • the EC50 value for mMCHlR-1-EGFP was nearly identical to that of the wild-type human short form receptor ( Figure 1).
  • the resulting fusion proteins exhibited functional activity comparable to that of the wild-type human MCHIR short form.
  • the EC50 value for the human short form/mouse species chimeric MCHlR-1-EGFP fusion protein was comparable to that of the corresponding wild-type human receptor, whereas, the human long form/mouse species chimeric MCHlR-1-EGFP fusion protein had an EC50 value approximately 7.5-fold higher than that of its corresponding wild-type control.
  • Wild-type CHO cells were transfected using SuperFect (Qiagen) and either mouse MCH-IR-EGFP or human short/mouse species chimeric MCH-IR- EGFP. Forty-eight hours after transfection, transfected cells were subjected to positive selection for approximately ten days in media containing G418. Following selection, MCH-IR-EGFP expressing CHO cells were bulk sorted by Fluorescence Assisted Cell Sorting (FACS) for one or two rounds on the basis of fluorescence intensity to increase the population of cells expressing EGFP. Following bulk sorts, individual clones of varying fluorescence intensities were isolated by FACS and expanded.
  • FACS Fluorescence Assisted Cell Sorting
  • Fluorometric Microvolume Assay Technology was initially employed to screen a large number of stable clones by whole cell binding with a fluorescently labeled MCH derivative (SymJz-MCH, PE Biosystems) to identify those clones with good specific binding windows.
  • a fluorescently labeled MCH derivative SymJz-MCH, PE Biosystems
  • Several clones exhibiting specific binding windows greater than 3 -fold were further evaluated for MCH binding with the SPA-based Binding Assay.
  • Cells from individual clones were dissociated in enzyme free dissociation media and cell membranes were prepared and subsequently tested for their ability to bind [ 125 I]Phe 13 Tyr 19 -MCH in the presence of human MCH.
  • CHO cell lines expressing either mouse MCH-IR-EGFP or human short/mouse species chimeric MCH-IR-EGFP ( Figure 4) displayed IC50 values with MCH that were indistinguishable from the corresponding IC50 values obtained with a CHO cell line expressing the wild-type human short isoform of MCH-IR.
  • CHO cell lines expressing either mouse MCH- IR-EGFP ( Figure 2) or human short/mouse species chimeric MCH-IR-EGFP ( Figure 3) displayed EC50 values with human MCH that were indistinguishable from the EC50 value obtained with a CHO cell line expressing the wild-type human short isoform of MCH-IR.
  • MCH-IR-EGFP fusion proteins were determined by confocal microscopy utilizing EGFP fluorescence as a marker for MCH-IR expression.

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Abstract

L'invention concerne des protéines de fusion et chimères du récepteur de l'hormone de concentration de la mélanine (MCH-R). Lesdites protéines chimères MCH-R comprennent une zone polypeptidique MCH-R constituée d'au moins deux zones polypeptidiques caractéristiques de MCH-R, que l'on rencontre dans différentes espèces. Les protéines de fusion MCH-R comprennent une zone polypeptidique MCH-R et une zone protéique fluorescente.
PCT/US2001/008071 2000-03-15 2001-03-14 Proteines chimeres et de fusion du recepteur de l'hormone de concentration de la melanine Ceased WO2001068706A1 (fr)

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JP2001567796A JP2003532388A (ja) 2000-03-15 2001-03-14 メラニン凝集ホルモン受容体キメラおよび融合タンパク質
EP01922374A EP1265926A1 (fr) 2000-03-15 2001-03-14 Proteines chimeres et de fusion du recepteur de l'hormone de concentration de la melanine
CA002402147A CA2402147A1 (fr) 2000-03-15 2001-03-14 Proteines chimeres et de fusion du recepteur de l'hormone de concentration de la melanine
US10/221,461 US7029878B2 (en) 2000-03-15 2001-03-14 Melanin concentrating hormone receptor chimeric and fusion proteins

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US7078187B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7078484B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
US7141391B2 (en) 2001-11-13 2006-11-28 Neurogen Corporation Monkey and canine melanin concentrating hormone receptors
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds
US7393655B2 (en) 1998-12-31 2008-07-01 H. Lundbeck A/S Methods of identifying melanin concentrating hormone receptor antagonists
EP2412729A3 (fr) * 2004-06-14 2012-07-25 Novo Nordisk A/S Purification de peptide au moyen d'une chromatographie d'affinité ionique de métal dur

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CN109813889A (zh) * 2017-11-18 2019-05-28 镇江亿特生物科技发展有限公司 检测孕酮的时间分辨荧光免疫层析定量检测试纸条

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BACHNER ET AL.: "Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1)", FEBS LETTERS, vol. 457, no. 3, 3 September 1999 (1999-09-03), pages 522 - 524, XP002942554 *
NELSON ET AL.: "Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion", ENDOCRINOLOGY, vol. 140, no. 2, February 1999 (1999-02-01), pages 950 - 957, XP002942552 *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7393655B2 (en) 1998-12-31 2008-07-01 H. Lundbeck A/S Methods of identifying melanin concentrating hormone receptor antagonists
EP1373545A4 (fr) * 2001-03-22 2005-07-20 Merck & Co Inc Souris deficientes en mch1r
US7259289B2 (en) 2001-03-22 2007-08-21 Merck & Co., Inc. MCH1R deficient mice
US7078187B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7078484B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
US7141391B2 (en) 2001-11-13 2006-11-28 Neurogen Corporation Monkey and canine melanin concentrating hormone receptors
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds
EP2412729A3 (fr) * 2004-06-14 2012-07-25 Novo Nordisk A/S Purification de peptide au moyen d'une chromatographie d'affinité ionique de métal dur

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