CA2562162A1 - Human g-protein chemokine receptor hdgnr10 - Google Patents
Human g-protein chemokine receptor hdgnr10 Download PDFInfo
- Publication number
- CA2562162A1 CA2562162A1 CA002562162A CA2562162A CA2562162A1 CA 2562162 A1 CA2562162 A1 CA 2562162A1 CA 002562162 A CA002562162 A CA 002562162A CA 2562162 A CA2562162 A CA 2562162A CA 2562162 A1 CA2562162 A1 CA 2562162A1
- Authority
- CA
- Canada
- Prior art keywords
- protein
- polypeptide
- polypeptides
- receptor
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000009410 Chemokine receptor Human genes 0.000 title claims abstract description 25
- 108050000299 Chemokine receptor Proteins 0.000 title claims abstract description 25
- 101000887490 Homo sapiens Guanine nucleotide-binding protein G(z) subunit alpha Proteins 0.000 title claims abstract 6
- 102000052301 human GNAZ Human genes 0.000 title claims abstract 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 155
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 153
- 229920001184 polypeptide Polymers 0.000 claims abstract description 152
- 238000000034 method Methods 0.000 claims abstract description 68
- 108090000623 proteins and genes Proteins 0.000 claims description 82
- 102000004169 proteins and genes Human genes 0.000 claims description 34
- 230000014509 gene expression Effects 0.000 claims description 30
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 5
- 208000038016 acute inflammation Diseases 0.000 claims description 3
- 230000006022 acute inflammation Effects 0.000 claims description 3
- 208000037976 chronic inflammation Diseases 0.000 claims description 3
- 230000006020 chronic inflammation Effects 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 3
- 238000011282 treatment Methods 0.000 claims description 3
- 230000009870 specific binding Effects 0.000 claims 2
- 238000012544 monitoring process Methods 0.000 claims 1
- 108020003175 receptors Proteins 0.000 abstract description 85
- 102000005962 receptors Human genes 0.000 abstract description 83
- 102000030782 GTP binding Human genes 0.000 abstract description 49
- 108091000058 GTP-Binding Proteins 0.000 abstract description 49
- 108091006027 G proteins Proteins 0.000 abstract description 43
- 150000007523 nucleic acids Chemical group 0.000 abstract description 22
- 239000005557 antagonist Substances 0.000 abstract description 8
- 230000035772 mutation Effects 0.000 abstract description 8
- 239000000556 agonist Substances 0.000 abstract description 5
- 238000010188 recombinant method Methods 0.000 abstract description 4
- 230000009452 underexpressoin Effects 0.000 abstract description 3
- 238000002405 diagnostic procedure Methods 0.000 abstract 1
- 230000002018 overexpression Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 94
- 108020004414 DNA Proteins 0.000 description 51
- 239000013598 vector Substances 0.000 description 48
- 102000040430 polynucleotide Human genes 0.000 description 43
- 108091033319 polynucleotide Proteins 0.000 description 43
- 239000002157 polynucleotide Substances 0.000 description 43
- 239000012634 fragment Substances 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 31
- 108091026890 Coding region Proteins 0.000 description 25
- 239000002299 complementary DNA Substances 0.000 description 22
- 239000003446 ligand Substances 0.000 description 22
- 150000001875 compounds Chemical class 0.000 description 20
- 230000027455 binding Effects 0.000 description 18
- 239000013612 plasmid Substances 0.000 description 18
- 241000282414 Homo sapiens Species 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 17
- 102000039446 nucleic acids Human genes 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 17
- 150000001413 amino acids Chemical class 0.000 description 16
- 108091034117 Oligonucleotide Proteins 0.000 description 15
- 230000004913 activation Effects 0.000 description 15
- 239000000523 sample Substances 0.000 description 15
- 239000013615 primer Substances 0.000 description 14
- 239000013604 expression vector Substances 0.000 description 13
- 238000012216 screening Methods 0.000 description 13
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000002773 nucleotide Substances 0.000 description 12
- 125000003729 nucleotide group Chemical group 0.000 description 12
- 230000001177 retroviral effect Effects 0.000 description 12
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 108091008146 restriction endonucleases Proteins 0.000 description 11
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 10
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 241000700605 Viruses Species 0.000 description 10
- 230000000875 corresponding effect Effects 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- 210000002950 fibroblast Anatomy 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 8
- 230000002068 genetic effect Effects 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 230000003612 virological effect Effects 0.000 description 8
- 102000019034 Chemokines Human genes 0.000 description 7
- 108010012236 Chemokines Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 210000000349 chromosome Anatomy 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 210000003527 eukaryotic cell Anatomy 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 238000004806 packaging method and process Methods 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 6
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 6
- 108091092195 Intron Proteins 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 230000002759 chromosomal effect Effects 0.000 description 6
- 238000013507 mapping Methods 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 230000010076 replication Effects 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 238000013519 translation Methods 0.000 description 6
- 241000701447 unidentified baculovirus Species 0.000 description 6
- 241000238631 Hexapoda Species 0.000 description 5
- 206010020751 Hypersensitivity Diseases 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 208000032839 leukemia Diseases 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 241000701161 unidentified adenovirus Species 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 102000004882 Lipase Human genes 0.000 description 4
- 108090001060 Lipase Proteins 0.000 description 4
- 239000004367 Lipase Substances 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 101710182846 Polyhedrin Proteins 0.000 description 4
- 102000006601 Thymidine Kinase Human genes 0.000 description 4
- 108020004440 Thymidine kinase Proteins 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 102000030621 adenylate cyclase Human genes 0.000 description 4
- 108060000200 adenylate cyclase Proteins 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 230000008827 biological function Effects 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 4
- -1 e.g. Proteins 0.000 description 4
- 210000002889 endothelial cell Anatomy 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 235000019421 lipase Nutrition 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- BYXHQQCXAJARLQ-ZLUOBGJFSA-N Ala-Ala-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O BYXHQQCXAJARLQ-ZLUOBGJFSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 239000003155 DNA primer Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 3
- 101710154606 Hemagglutinin Proteins 0.000 description 3
- 241000880493 Leptailurus serval Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 3
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 3
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 3
- 108091000080 Phosphotransferase Proteins 0.000 description 3
- 101710176177 Protein A56 Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 108020005091 Replication Origin Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- BIBYEFRASCNLAA-CDMKHQONSA-N Thr-Phe-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 BIBYEFRASCNLAA-CDMKHQONSA-N 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 208000026935 allergic disease Diseases 0.000 description 3
- 230000007815 allergy Effects 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 210000003050 axon Anatomy 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 210000001072 colon Anatomy 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 229960001340 histamine Drugs 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229960004452 methionine Drugs 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 3
- 102000020233 phosphotransferase Human genes 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000035939 shock Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000014621 translational initiation Effects 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 108010073969 valyllysine Proteins 0.000 description 3
- PWYFCPCBOYMOGB-LKTVYLICSA-N Ala-Gln-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N PWYFCPCBOYMOGB-LKTVYLICSA-N 0.000 description 2
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-N 0.000 description 2
- 206010002198 Anaphylactic reaction Diseases 0.000 description 2
- 241000272517 Anseriformes Species 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- JSLGXODUIAFWCF-WDSKDSINSA-N Arg-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O JSLGXODUIAFWCF-WDSKDSINSA-N 0.000 description 2
- HJAICMSAKODKRF-GUBZILKMSA-N Arg-Cys-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O HJAICMSAKODKRF-GUBZILKMSA-N 0.000 description 2
- XRNXPIGJPQHCPC-RCWTZXSCSA-N Arg-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)O)C(O)=O XRNXPIGJPQHCPC-RCWTZXSCSA-N 0.000 description 2
- SPCONPVIDFMDJI-QSFUFRPTSA-N Asn-Ile-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O SPCONPVIDFMDJI-QSFUFRPTSA-N 0.000 description 2
- ILJQISGMGXRZQQ-IHRRRGAJSA-N Asp-Arg-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ILJQISGMGXRZQQ-IHRRRGAJSA-N 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 208000018240 Bone Marrow Failure disease Diseases 0.000 description 2
- 206010065553 Bone marrow failure Diseases 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- OABOXRPGTFRBFZ-IMJSIDKUSA-N Cys-Cys Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(O)=O OABOXRPGTFRBFZ-IMJSIDKUSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- YTRBQAQSUDSIQE-FHWLQOOXSA-N Glu-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 YTRBQAQSUDSIQE-FHWLQOOXSA-N 0.000 description 2
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 241000257303 Hymenoptera Species 0.000 description 2
- 206010048643 Hypereosinophilic syndrome Diseases 0.000 description 2
- QIHJTGSVGIPHIW-QSFUFRPTSA-N Ile-Asn-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N QIHJTGSVGIPHIW-QSFUFRPTSA-N 0.000 description 2
- TVYWVSJGSHQWMT-AJNGGQMLSA-N Ile-Leu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N TVYWVSJGSHQWMT-AJNGGQMLSA-N 0.000 description 2
- UAELWXJFLZBKQS-WHOFXGATSA-N Ile-Phe-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O UAELWXJFLZBKQS-WHOFXGATSA-N 0.000 description 2
- DLEBSGAVWRPTIX-PEDHHIEDSA-N Ile-Val-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)[C@@H](C)CC DLEBSGAVWRPTIX-PEDHHIEDSA-N 0.000 description 2
- 108090001007 Interleukin-8 Proteins 0.000 description 2
- 102000004890 Interleukin-8 Human genes 0.000 description 2
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 241000272168 Laridae Species 0.000 description 2
- KFKWRHQBZQICHA-STQMWFEESA-N Leu-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 2
- XOEDPXDZJHBQIX-ULQDDVLXSA-N Leu-Val-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XOEDPXDZJHBQIX-ULQDDVLXSA-N 0.000 description 2
- QCZYYEFXOBKCNQ-STQMWFEESA-N Lys-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QCZYYEFXOBKCNQ-STQMWFEESA-N 0.000 description 2
- PNDCUTDWYVKBHX-IHRRRGAJSA-N Met-Asp-Tyr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 PNDCUTDWYVKBHX-IHRRRGAJSA-N 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 208000037581 Persistent Infection Diseases 0.000 description 2
- ODGNUUUDJONJSC-UFYCRDLUSA-N Phe-Pro-Tyr Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O ODGNUUUDJONJSC-UFYCRDLUSA-N 0.000 description 2
- FGWUALWGCZJQDJ-URLPEUOOSA-N Phe-Thr-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGWUALWGCZJQDJ-URLPEUOOSA-N 0.000 description 2
- XALFIVXGQUEGKV-JSGCOSHPSA-N Phe-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 XALFIVXGQUEGKV-JSGCOSHPSA-N 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 102100030304 Platelet factor 4 Human genes 0.000 description 2
- 108090000778 Platelet factor 4 Proteins 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- FEPSEIDIPBMIOS-QXEWZRGKSA-N Pro-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 FEPSEIDIPBMIOS-QXEWZRGKSA-N 0.000 description 2
- RYJRPPUATSKNAY-STECZYCISA-N Pro-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@@H]2CCCN2 RYJRPPUATSKNAY-STECZYCISA-N 0.000 description 2
- YYARMJSFDLIDFS-FKBYEOEOSA-N Pro-Phe-Trp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O YYARMJSFDLIDFS-FKBYEOEOSA-N 0.000 description 2
- 201000004681 Psoriasis Diseases 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 241000725643 Respiratory syncytial virus Species 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 201000010001 Silicosis Diseases 0.000 description 2
- BECPPKYKPSRKCP-ZDLURKLDSA-N Thr-Glu Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O BECPPKYKPSRKCP-ZDLURKLDSA-N 0.000 description 2
- BQBCIBCLXBKYHW-CSMHCCOUSA-N Thr-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@@H]([NH3+])[C@@H](C)O BQBCIBCLXBKYHW-CSMHCCOUSA-N 0.000 description 2
- PZSDPRBZINDEJV-HTUGSXCWSA-N Thr-Phe-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O PZSDPRBZINDEJV-HTUGSXCWSA-N 0.000 description 2
- 102000014384 Type C Phospholipases Human genes 0.000 description 2
- 108010079194 Type C Phospholipases Proteins 0.000 description 2
- VCXWRWYFJLXITF-AUTRQRHGSA-N Tyr-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 VCXWRWYFJLXITF-AUTRQRHGSA-N 0.000 description 2
- FASACHWGQBNSRO-ZEWNOJEFSA-N Tyr-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CC2=CC=C(C=C2)O)N FASACHWGQBNSRO-ZEWNOJEFSA-N 0.000 description 2
- ITDWWLTTWRRLCC-KJEVXHAQSA-N Tyr-Thr-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 ITDWWLTTWRRLCC-KJEVXHAQSA-N 0.000 description 2
- WFENBJPLZMPVAX-XVKPBYJWSA-N Val-Gly-Glu Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O WFENBJPLZMPVAX-XVKPBYJWSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 108010045023 alanyl-prolyl-tyrosine Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000036783 anaphylactic response Effects 0.000 description 2
- 208000003455 anaphylaxis Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000036523 atherogenesis Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 108010004073 cysteinylcysteine Proteins 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 108091008039 hormone receptors Proteins 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 description 2
- 229940096397 interleukin-8 Drugs 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 210000002510 keratinocyte Anatomy 0.000 description 2
- 101150109249 lacI gene Proteins 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 108010038320 lysylphenylalanine Proteins 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 210000000287 oocyte Anatomy 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 108010024607 phenylalanylalanine Proteins 0.000 description 2
- 108010073101 phenylalanylleucine Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 108010070643 prolylglutamic acid Proteins 0.000 description 2
- 150000003180 prostaglandins Chemical class 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 201000000306 sarcoidosis Diseases 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 description 1
- OJHZNMVJJKMFGX-RNWHKREASA-N (4r,4ar,7ar,12bs)-9-methoxy-3-methyl-1,2,4,4a,5,6,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one;2,3-dihydroxybutanedioic acid Chemical compound OC(=O)C(O)C(O)C(O)=O.O=C([C@@H]1O2)CC[C@H]3[C@]4([H])N(C)CC[C@]13C1=C2C(OC)=CC=C1C4 OJHZNMVJJKMFGX-RNWHKREASA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- SCPRYBYMKVYVND-UHFFFAOYSA-N 2-[[2-[[1-(2-amino-4-methylpentanoyl)pyrrolidine-2-carbonyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoic acid Chemical compound CC(C)CC(N)C(=O)N1CCCC1C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(O)=O SCPRYBYMKVYVND-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 241000484419 Aedia Species 0.000 description 1
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 1
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 1
- OARAZORWIMYUPO-FXQIFTODSA-N Ala-Met-Cys Chemical compound CSCC[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CS)C(O)=O OARAZORWIMYUPO-FXQIFTODSA-N 0.000 description 1
- CQJHFKKGZXKZBC-BPNCWPANSA-N Ala-Pro-Tyr Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CQJHFKKGZXKZBC-BPNCWPANSA-N 0.000 description 1
- IYKVSFNGSWTTNZ-GUBZILKMSA-N Ala-Val-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IYKVSFNGSWTTNZ-GUBZILKMSA-N 0.000 description 1
- 108020004491 Antisense DNA Proteins 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- IJPNNYWHXGADJG-GUBZILKMSA-N Arg-Ala-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O IJPNNYWHXGADJG-GUBZILKMSA-N 0.000 description 1
- OTUQSEPIIVBYEM-IHRRRGAJSA-N Arg-Asn-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OTUQSEPIIVBYEM-IHRRRGAJSA-N 0.000 description 1
- BNODVYXZAAXSHW-IUCAKERBSA-N Arg-His Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 BNODVYXZAAXSHW-IUCAKERBSA-N 0.000 description 1
- XUGATJVGQUGQKY-ULQDDVLXSA-N Arg-Lys-Phe Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XUGATJVGQUGQKY-ULQDDVLXSA-N 0.000 description 1
- BSGSDLYGGHGMND-IHRRRGAJSA-N Arg-Phe-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N BSGSDLYGGHGMND-IHRRRGAJSA-N 0.000 description 1
- IJYZHIOOBGIINM-WDSKDSINSA-N Arg-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N IJYZHIOOBGIINM-WDSKDSINSA-N 0.000 description 1
- VRTWYUYCJGNFES-CIUDSAMLSA-N Arg-Ser-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O VRTWYUYCJGNFES-CIUDSAMLSA-N 0.000 description 1
- FRBAHXABMQXSJQ-FXQIFTODSA-N Arg-Ser-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FRBAHXABMQXSJQ-FXQIFTODSA-N 0.000 description 1
- CGWVCWFQGXOUSJ-ULQDDVLXSA-N Arg-Tyr-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O CGWVCWFQGXOUSJ-ULQDDVLXSA-N 0.000 description 1
- YNSCBOUZTAGIGO-ZLUOBGJFSA-N Asn-Asn-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N)C(=O)N YNSCBOUZTAGIGO-ZLUOBGJFSA-N 0.000 description 1
- LUVODTFFSXVOAG-ACZMJKKPSA-N Asn-Cys-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)N)N LUVODTFFSXVOAG-ACZMJKKPSA-N 0.000 description 1
- SPIPSJXLZVTXJL-ZLUOBGJFSA-N Asn-Cys-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O SPIPSJXLZVTXJL-ZLUOBGJFSA-N 0.000 description 1
- GYOHQKJEQQJBOY-QEJZJMRPSA-N Asn-Glu-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)N)N GYOHQKJEQQJBOY-QEJZJMRPSA-N 0.000 description 1
- HYQYLOSCICEYTR-YUMQZZPRSA-N Asn-Gly-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O HYQYLOSCICEYTR-YUMQZZPRSA-N 0.000 description 1
- JLNFZLNDHONLND-GARJFASQSA-N Asn-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N JLNFZLNDHONLND-GARJFASQSA-N 0.000 description 1
- AEZCCDMZZJOGII-DCAQKATOSA-N Asn-Met-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O AEZCCDMZZJOGII-DCAQKATOSA-N 0.000 description 1
- OMSMPWHEGLNQOD-UWVGGRQHSA-N Asn-Phe Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OMSMPWHEGLNQOD-UWVGGRQHSA-N 0.000 description 1
- NJSNXIOKBHPFMB-GMOBBJLQSA-N Asn-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)N)N NJSNXIOKBHPFMB-GMOBBJLQSA-N 0.000 description 1
- VBKIFHUVGLOJKT-FKZODXBYSA-N Asn-Thr Chemical compound C[C@@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)N)O VBKIFHUVGLOJKT-FKZODXBYSA-N 0.000 description 1
- XIDSGDJNUJRUHE-VEVYYDQMSA-N Asn-Thr-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(O)=O XIDSGDJNUJRUHE-VEVYYDQMSA-N 0.000 description 1
- LRCIOEVFVGXZKB-BZSNNMDCSA-N Asn-Tyr-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LRCIOEVFVGXZKB-BZSNNMDCSA-N 0.000 description 1
- NYQHSUGFEWDWPD-ACZMJKKPSA-N Asp-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N NYQHSUGFEWDWPD-ACZMJKKPSA-N 0.000 description 1
- HKEZZWQWXWGASX-KKUMJFAQSA-N Asp-Leu-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 HKEZZWQWXWGASX-KKUMJFAQSA-N 0.000 description 1
- QJHOOKBAHRJPPX-QWRGUYRKSA-N Asp-Phe-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 QJHOOKBAHRJPPX-QWRGUYRKSA-N 0.000 description 1
- AWPWHMVCSISSQK-QWRGUYRKSA-N Asp-Tyr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O AWPWHMVCSISSQK-QWRGUYRKSA-N 0.000 description 1
- 241000201370 Autographa californica nucleopolyhedrovirus Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 102100021277 Beta-secretase 2 Human genes 0.000 description 1
- 101710150190 Beta-secretase 2 Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical group [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 101100108211 Candida albicans (strain SC5314 / ATCC MYA-2876) ADF1 gene Proteins 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 108091033380 Coding strand Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 1
- SMYXEYRYCLIPIL-ZLUOBGJFSA-N Cys-Cys-Cys Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O SMYXEYRYCLIPIL-ZLUOBGJFSA-N 0.000 description 1
- YHDXIZKDOIWPBW-WHFBIAKZSA-N Cys-Gln Chemical compound SC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O YHDXIZKDOIWPBW-WHFBIAKZSA-N 0.000 description 1
- LKUCSUGWHYVYLP-GHCJXIJMSA-N Cys-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CS)N LKUCSUGWHYVYLP-GHCJXIJMSA-N 0.000 description 1
- KGIHMGPYGXBYJJ-SRVKXCTJSA-N Cys-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CS KGIHMGPYGXBYJJ-SRVKXCTJSA-N 0.000 description 1
- HMWBPUDETPKSSS-DCAQKATOSA-N Cys-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CS)N)C(=O)N[C@@H](CCCCN)C(=O)O HMWBPUDETPKSSS-DCAQKATOSA-N 0.000 description 1
- DSTWKJOBKSMVCV-UWVGGRQHSA-N Cys-Tyr Chemical compound SC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DSTWKJOBKSMVCV-UWVGGRQHSA-N 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 108010090461 DFG peptide Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000283715 Damaliscus lunatus Species 0.000 description 1
- 102000015554 Dopamine receptor Human genes 0.000 description 1
- 108050004812 Dopamine receptor Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101150104377 Ecel1 gene Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 101001039702 Escherichia coli (strain K12) Methyl-accepting chemotaxis protein I Proteins 0.000 description 1
- 241001596967 Escherichia coli M15 Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 241000155335 Foza Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- XXLBHPPXDUWYAG-XQXXSGGOSA-N Gln-Ala-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XXLBHPPXDUWYAG-XQXXSGGOSA-N 0.000 description 1
- JEFZIKRIDLHOIF-BYPYZUCNSA-N Gln-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(O)=O JEFZIKRIDLHOIF-BYPYZUCNSA-N 0.000 description 1
- OAOOXBSVCJEIFY-QAETUUGQSA-N Gln-Leu-Leu-Pro Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O OAOOXBSVCJEIFY-QAETUUGQSA-N 0.000 description 1
- CLSDNFWKGFJIBZ-YUMQZZPRSA-N Gln-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(N)=O CLSDNFWKGFJIBZ-YUMQZZPRSA-N 0.000 description 1
- PDXIOFXRBVDSHD-JBACZVJFSA-N Gln-Phe-Trp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)NC(=O)[C@H](CCC(=O)N)N PDXIOFXRBVDSHD-JBACZVJFSA-N 0.000 description 1
- HNAUFGBKJLTWQE-IFFSRLJSSA-N Gln-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)N)N)O HNAUFGBKJLTWQE-IFFSRLJSSA-N 0.000 description 1
- IRDASPPCLZIERZ-XHNCKOQMSA-N Glu-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N IRDASPPCLZIERZ-XHNCKOQMSA-N 0.000 description 1
- AVZHGSCDKIQZPQ-CIUDSAMLSA-N Glu-Arg-Ala Chemical compound C[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AVZHGSCDKIQZPQ-CIUDSAMLSA-N 0.000 description 1
- QJCKNLPMTPXXEM-AUTRQRHGSA-N Glu-Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O QJCKNLPMTPXXEM-AUTRQRHGSA-N 0.000 description 1
- LRPXYSGPOBVBEH-IUCAKERBSA-N Glu-Gly-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O LRPXYSGPOBVBEH-IUCAKERBSA-N 0.000 description 1
- ZHNHJYYFCGUZNQ-KBIXCLLPSA-N Glu-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O ZHNHJYYFCGUZNQ-KBIXCLLPSA-N 0.000 description 1
- HLYCMRDRWGSTPZ-CIUDSAMLSA-N Glu-Pro-Cys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CS)C(=O)O HLYCMRDRWGSTPZ-CIUDSAMLSA-N 0.000 description 1
- WIKMTDVSCUJIPJ-CIUDSAMLSA-N Glu-Ser-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WIKMTDVSCUJIPJ-CIUDSAMLSA-N 0.000 description 1
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- FMNHBTKMRFVGRO-FOHZUACHSA-N Gly-Asn-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CN FMNHBTKMRFVGRO-FOHZUACHSA-N 0.000 description 1
- YZACQYVWLCQWBT-BQBZGAKWSA-N Gly-Cys-Arg Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O YZACQYVWLCQWBT-BQBZGAKWSA-N 0.000 description 1
- XMPXVJIDADUOQB-RCOVLWMOSA-N Gly-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C([O-])=O)NC(=O)CNC(=O)C[NH3+] XMPXVJIDADUOQB-RCOVLWMOSA-N 0.000 description 1
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 1
- FEUPVVCGQLNXNP-IRXDYDNUSA-N Gly-Phe-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FEUPVVCGQLNXNP-IRXDYDNUSA-N 0.000 description 1
- VDCRBJACQKOSMS-JSGCOSHPSA-N Gly-Phe-Val Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O VDCRBJACQKOSMS-JSGCOSHPSA-N 0.000 description 1
- OCRQUYDOYKCOQG-IRXDYDNUSA-N Gly-Tyr-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 OCRQUYDOYKCOQG-IRXDYDNUSA-N 0.000 description 1
- GJHWILMUOANXTG-WPRPVWTQSA-N Gly-Val-Arg Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GJHWILMUOANXTG-WPRPVWTQSA-N 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- HXKZJLWGSWQKEA-LSJOCFKGSA-N His-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CN=CN1 HXKZJLWGSWQKEA-LSJOCFKGSA-N 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- CSTDQOOBZBAJKE-BWAGICSOSA-N His-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N)O CSTDQOOBZBAJKE-BWAGICSOSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000897480 Homo sapiens C-C motif chemokine 2 Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 241000282620 Hylobates sp. Species 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- DPTBVFUDCPINIP-JURCDPSOSA-N Ile-Ala-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 DPTBVFUDCPINIP-JURCDPSOSA-N 0.000 description 1
- UKTUOMWSJPXODT-GUDRVLHUSA-N Ile-Asn-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N UKTUOMWSJPXODT-GUDRVLHUSA-N 0.000 description 1
- LEDRIAHEWDJRMF-CFMVVWHZSA-N Ile-Asn-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 LEDRIAHEWDJRMF-CFMVVWHZSA-N 0.000 description 1
- VCYVLFAWCJRXFT-HJPIBITLSA-N Ile-Cys-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N VCYVLFAWCJRXFT-HJPIBITLSA-N 0.000 description 1
- NZOCIWKZUVUNDW-ZKWXMUAHSA-N Ile-Gly-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O NZOCIWKZUVUNDW-ZKWXMUAHSA-N 0.000 description 1
- NYEYYMLUABXDMC-NHCYSSNCSA-N Ile-Gly-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)O)N NYEYYMLUABXDMC-NHCYSSNCSA-N 0.000 description 1
- BCVIOZZGJNOEQS-XKNYDFJKSA-N Ile-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(O)=O)[C@@H](C)CC BCVIOZZGJNOEQS-XKNYDFJKSA-N 0.000 description 1
- TWPSALMCEHCIOY-YTFOTSKYSA-N Ile-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)O)N TWPSALMCEHCIOY-YTFOTSKYSA-N 0.000 description 1
- JWBXCSQZLLIOCI-GUBZILKMSA-N Ile-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(O)=O)CC(C)C JWBXCSQZLLIOCI-GUBZILKMSA-N 0.000 description 1
- GAZGFPOZOLEYAJ-YTFOTSKYSA-N Ile-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N GAZGFPOZOLEYAJ-YTFOTSKYSA-N 0.000 description 1
- HPCFRQWLTRDGHT-AJNGGQMLSA-N Ile-Leu-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O HPCFRQWLTRDGHT-AJNGGQMLSA-N 0.000 description 1
- CIDLJWVDMNDKPT-FIRPJDEBSA-N Ile-Phe-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N CIDLJWVDMNDKPT-FIRPJDEBSA-N 0.000 description 1
- VEPIBPGLTLPBDW-URLPEUOOSA-N Ile-Phe-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N VEPIBPGLTLPBDW-URLPEUOOSA-N 0.000 description 1
- XOZOSAUOGRPCES-STECZYCISA-N Ile-Pro-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XOZOSAUOGRPCES-STECZYCISA-N 0.000 description 1
- HXIDVIFHRYRXLZ-NAKRPEOUSA-N Ile-Ser-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)O)N HXIDVIFHRYRXLZ-NAKRPEOUSA-N 0.000 description 1
- DRCKHKZYDLJYFQ-YWIQKCBGSA-N Ile-Thr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DRCKHKZYDLJYFQ-YWIQKCBGSA-N 0.000 description 1
- DTPGSUQHUMELQB-GVARAGBVSA-N Ile-Tyr-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 DTPGSUQHUMELQB-GVARAGBVSA-N 0.000 description 1
- GVEODXUBBFDBPW-MGHWNKPDSA-N Ile-Tyr-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 GVEODXUBBFDBPW-MGHWNKPDSA-N 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000002397 Kinins Human genes 0.000 description 1
- 108010093008 Kinins Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- QLROSWPKSBORFJ-BQBZGAKWSA-N L-Prolyl-L-glutamic acid Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 QLROSWPKSBORFJ-BQBZGAKWSA-N 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- QPRQGENIBFLVEB-BJDJZHNGSA-N Leu-Ala-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O QPRQGENIBFLVEB-BJDJZHNGSA-N 0.000 description 1
- DUBAVOVZNZKEQQ-AVGNSLFASA-N Leu-Arg-Val Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CCCN=C(N)N DUBAVOVZNZKEQQ-AVGNSLFASA-N 0.000 description 1
- JKGHDYGZRDWHGA-SRVKXCTJSA-N Leu-Asn-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JKGHDYGZRDWHGA-SRVKXCTJSA-N 0.000 description 1
- PJYSOYLLTJKZHC-GUBZILKMSA-N Leu-Asp-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O PJYSOYLLTJKZHC-GUBZILKMSA-N 0.000 description 1
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 1
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 1
- QPXBPQUGXHURGP-UWVGGRQHSA-N Leu-Gly-Met Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CCSC)C(=O)O)N QPXBPQUGXHURGP-UWVGGRQHSA-N 0.000 description 1
- USLNHQZCDQJBOV-ZPFDUUQYSA-N Leu-Ile-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O USLNHQZCDQJBOV-ZPFDUUQYSA-N 0.000 description 1
- ZALAVHVPPOHAOL-XUXIUFHCSA-N Leu-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(C)C)N ZALAVHVPPOHAOL-XUXIUFHCSA-N 0.000 description 1
- QLDHBYRUNQZIJQ-DKIMLUQUSA-N Leu-Ile-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QLDHBYRUNQZIJQ-DKIMLUQUSA-N 0.000 description 1
- LIINDKYIGYTDLG-PPCPHDFISA-N Leu-Ile-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LIINDKYIGYTDLG-PPCPHDFISA-N 0.000 description 1
- IAJFFZORSWOZPQ-SRVKXCTJSA-N Leu-Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IAJFFZORSWOZPQ-SRVKXCTJSA-N 0.000 description 1
- UBZGNBKMIJHOHL-BZSNNMDCSA-N Leu-Leu-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 UBZGNBKMIJHOHL-BZSNNMDCSA-N 0.000 description 1
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 1
- DCGXHWINSHEPIR-SRVKXCTJSA-N Leu-Lys-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)O)N DCGXHWINSHEPIR-SRVKXCTJSA-N 0.000 description 1
- MAXILRZVORNXBE-PMVMPFDFSA-N Leu-Phe-Trp Chemical compound C([C@H](NC(=O)[C@@H](N)CC(C)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 MAXILRZVORNXBE-PMVMPFDFSA-N 0.000 description 1
- KWLWZYMNUZJKMZ-IHRRRGAJSA-N Leu-Pro-Leu Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O KWLWZYMNUZJKMZ-IHRRRGAJSA-N 0.000 description 1
- ICYRCNICGBJLGM-HJGDQZAQSA-N Leu-Thr-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O ICYRCNICGBJLGM-HJGDQZAQSA-N 0.000 description 1
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 1
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 1
- VUBIPAHVHMZHCM-KKUMJFAQSA-N Leu-Tyr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 VUBIPAHVHMZHCM-KKUMJFAQSA-N 0.000 description 1
- FMFNIDICDKEMOE-XUXIUFHCSA-N Leu-Val-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FMFNIDICDKEMOE-XUXIUFHCSA-N 0.000 description 1
- RVOMPSJXSRPFJT-DCAQKATOSA-N Lys-Ala-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RVOMPSJXSRPFJT-DCAQKATOSA-N 0.000 description 1
- NPBGTPKLVJEOBE-IUCAKERBSA-N Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N NPBGTPKLVJEOBE-IUCAKERBSA-N 0.000 description 1
- BRSGXFITDXFMFF-IHRRRGAJSA-N Lys-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)N BRSGXFITDXFMFF-IHRRRGAJSA-N 0.000 description 1
- JPNRPAJITHRXRH-BQBZGAKWSA-N Lys-Asn Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC(N)=O JPNRPAJITHRXRH-BQBZGAKWSA-N 0.000 description 1
- YVSHZSUKQHNDHD-KKUMJFAQSA-N Lys-Asn-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N YVSHZSUKQHNDHD-KKUMJFAQSA-N 0.000 description 1
- CFVQPNSCQMKDPB-CIUDSAMLSA-N Lys-Cys-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)O)N CFVQPNSCQMKDPB-CIUDSAMLSA-N 0.000 description 1
- MLLKLNYPZRDIQG-GUBZILKMSA-N Lys-Cys-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N MLLKLNYPZRDIQG-GUBZILKMSA-N 0.000 description 1
- YVMQJGWLHRWMDF-MNXVOIDGSA-N Lys-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N YVMQJGWLHRWMDF-MNXVOIDGSA-N 0.000 description 1
- UGTZHPSKYRIGRJ-YUMQZZPRSA-N Lys-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O UGTZHPSKYRIGRJ-YUMQZZPRSA-N 0.000 description 1
- GCMWRRQAKQXDED-IUCAKERBSA-N Lys-Glu-Gly Chemical compound [NH3+]CCCC[C@H]([NH3+])C(=O)N[C@@H](CCC([O-])=O)C(=O)NCC([O-])=O GCMWRRQAKQXDED-IUCAKERBSA-N 0.000 description 1
- HGNRJCINZYHNOU-LURJTMIESA-N Lys-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(O)=O HGNRJCINZYHNOU-LURJTMIESA-N 0.000 description 1
- XIZQPFCRXLUNMK-BZSNNMDCSA-N Lys-Leu-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCCCN)N XIZQPFCRXLUNMK-BZSNNMDCSA-N 0.000 description 1
- NVGBPTNZLWRQSY-UWVGGRQHSA-N Lys-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN NVGBPTNZLWRQSY-UWVGGRQHSA-N 0.000 description 1
- TWPCWKVOZDUYAA-KKUMJFAQSA-N Lys-Phe-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O TWPCWKVOZDUYAA-KKUMJFAQSA-N 0.000 description 1
- YSZNURNVYFUEHC-BQBZGAKWSA-N Lys-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(O)=O YSZNURNVYFUEHC-BQBZGAKWSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- NCFZHKMKRCYQBJ-CIUDSAMLSA-N Met-Cys-Gln Chemical compound CSCC[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N NCFZHKMKRCYQBJ-CIUDSAMLSA-N 0.000 description 1
- ODFBIJXEWPWSAN-CYDGBPFRSA-N Met-Ile-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O ODFBIJXEWPWSAN-CYDGBPFRSA-N 0.000 description 1
- KPVLLNDCBYXKNV-CYDGBPFRSA-N Met-Val-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KPVLLNDCBYXKNV-CYDGBPFRSA-N 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241000713862 Moloney murine sarcoma virus Species 0.000 description 1
- 101001043818 Mus musculus Interleukin-31 receptor subunit alpha Proteins 0.000 description 1
- 101000969137 Mus musculus Metallothionein-1 Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000713883 Myeloproliferative sarcoma virus Species 0.000 description 1
- MDSUKZSLOATHMH-UHFFFAOYSA-N N-L-leucyl-L-valine Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(O)=O MDSUKZSLOATHMH-UHFFFAOYSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102000010175 Opsin Human genes 0.000 description 1
- 108050001704 Opsin Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- MIDZLCFIAINOQN-WPRPVWTQSA-N Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 MIDZLCFIAINOQN-WPRPVWTQSA-N 0.000 description 1
- BBDSZDHUCPSYAC-QEJZJMRPSA-N Phe-Ala-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O BBDSZDHUCPSYAC-QEJZJMRPSA-N 0.000 description 1
- UHRNIXJAGGLKHP-DLOVCJGASA-N Phe-Ala-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O UHRNIXJAGGLKHP-DLOVCJGASA-N 0.000 description 1
- IWRZUGHCHFZYQZ-UFYCRDLUSA-N Phe-Arg-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 IWRZUGHCHFZYQZ-UFYCRDLUSA-N 0.000 description 1
- CUMXHKAOHNWRFQ-BZSNNMDCSA-N Phe-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 CUMXHKAOHNWRFQ-BZSNNMDCSA-N 0.000 description 1
- KLAONOISLHWJEE-QWRGUYRKSA-N Phe-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KLAONOISLHWJEE-QWRGUYRKSA-N 0.000 description 1
- IILUKIJNFMUBNF-IHRRRGAJSA-N Phe-Gln-Gln Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O IILUKIJNFMUBNF-IHRRRGAJSA-N 0.000 description 1
- WYPVCIACUMJRIB-JYJNAYRXSA-N Phe-Gln-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N WYPVCIACUMJRIB-JYJNAYRXSA-N 0.000 description 1
- NKLDZIPTGKBDBB-HTUGSXCWSA-N Phe-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N)O NKLDZIPTGKBDBB-HTUGSXCWSA-N 0.000 description 1
- WPTYDQPGBMDUBI-QWRGUYRKSA-N Phe-Gly-Asn Chemical compound N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O WPTYDQPGBMDUBI-QWRGUYRKSA-N 0.000 description 1
- APJPXSFJBMMOLW-KBPBESRZSA-N Phe-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 APJPXSFJBMMOLW-KBPBESRZSA-N 0.000 description 1
- BVHFFNYBKRTSIU-MEYUZBJRSA-N Phe-His-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BVHFFNYBKRTSIU-MEYUZBJRSA-N 0.000 description 1
- RFCVXVPWSPOMFJ-STQMWFEESA-N Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 RFCVXVPWSPOMFJ-STQMWFEESA-N 0.000 description 1
- VHDNDCPMHQMXIR-IHRRRGAJSA-N Phe-Met-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CC1=CC=CC=C1 VHDNDCPMHQMXIR-IHRRRGAJSA-N 0.000 description 1
- GKZIWHRNKRBEOH-HOTGVXAUSA-N Phe-Phe Chemical compound C([C@H]([NH3+])C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)C1=CC=CC=C1 GKZIWHRNKRBEOH-HOTGVXAUSA-N 0.000 description 1
- FENSZYFJQOFSQR-FIRPJDEBSA-N Phe-Phe-Ile Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FENSZYFJQOFSQR-FIRPJDEBSA-N 0.000 description 1
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 1
- NYQBYASWHVRESG-MIMYLULJSA-N Phe-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 NYQBYASWHVRESG-MIMYLULJSA-N 0.000 description 1
- LTAWNJXSRUCFAN-UNQGMJICSA-N Phe-Thr-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LTAWNJXSRUCFAN-UNQGMJICSA-N 0.000 description 1
- BPIFSOUEUYDJRM-DCPHZVHLSA-N Phe-Trp-Ala Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(O)=O)C1=CC=CC=C1 BPIFSOUEUYDJRM-DCPHZVHLSA-N 0.000 description 1
- LKRUQZQZMXMKEQ-SFJXLCSZSA-N Phe-Trp-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LKRUQZQZMXMKEQ-SFJXLCSZSA-N 0.000 description 1
- 102000011420 Phospholipase D Human genes 0.000 description 1
- 108090000553 Phospholipase D Proteins 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 1
- OGRYXQOUFHAMPI-DCAQKATOSA-N Pro-Cys-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O OGRYXQOUFHAMPI-DCAQKATOSA-N 0.000 description 1
- ZKQOUHVVXABNDG-IUCAKERBSA-N Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 ZKQOUHVVXABNDG-IUCAKERBSA-N 0.000 description 1
- CPRLKHJUFAXVTD-ULQDDVLXSA-N Pro-Leu-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CPRLKHJUFAXVTD-ULQDDVLXSA-N 0.000 description 1
- RWCOTTLHDJWHRS-YUMQZZPRSA-N Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RWCOTTLHDJWHRS-YUMQZZPRSA-N 0.000 description 1
- STGVYUTZKGPRCI-GUBZILKMSA-N Pro-Val-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 STGVYUTZKGPRCI-GUBZILKMSA-N 0.000 description 1
- 102000003923 Protein Kinase C Human genes 0.000 description 1
- 108090000315 Protein Kinase C Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 208000028017 Psychotic disease Diseases 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 102000004330 Rhodopsin Human genes 0.000 description 1
- 108090000820 Rhodopsin Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 101100379247 Salmo trutta apoa1 gene Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- FIDMVVBUOCMMJG-CIUDSAMLSA-N Ser-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO FIDMVVBUOCMMJG-CIUDSAMLSA-N 0.000 description 1
- UJTZHGHXJKIAOS-WHFBIAKZSA-N Ser-Gln Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O UJTZHGHXJKIAOS-WHFBIAKZSA-N 0.000 description 1
- VMVNCJDKFOQOHM-GUBZILKMSA-N Ser-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CO)N VMVNCJDKFOQOHM-GUBZILKMSA-N 0.000 description 1
- VDVYTKZBMFADQH-AVGNSLFASA-N Ser-Gln-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 VDVYTKZBMFADQH-AVGNSLFASA-N 0.000 description 1
- HVKMTOIAYDOJPL-NRPADANISA-N Ser-Gln-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O HVKMTOIAYDOJPL-NRPADANISA-N 0.000 description 1
- UFKPDBLKLOBMRH-XHNCKOQMSA-N Ser-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)C(=O)O UFKPDBLKLOBMRH-XHNCKOQMSA-N 0.000 description 1
- WOUIMBGNEUWXQG-VKHMYHEASA-N Ser-Gly Chemical compound OC[C@H](N)C(=O)NCC(O)=O WOUIMBGNEUWXQG-VKHMYHEASA-N 0.000 description 1
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 1
- IXZHZUGGKLRHJD-DCAQKATOSA-N Ser-Leu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O IXZHZUGGKLRHJD-DCAQKATOSA-N 0.000 description 1
- RXSWQCATLWVDLI-XGEHTFHBSA-N Ser-Met-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RXSWQCATLWVDLI-XGEHTFHBSA-N 0.000 description 1
- GZGFSPWOMUKKCV-NAKRPEOUSA-N Ser-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO GZGFSPWOMUKKCV-NAKRPEOUSA-N 0.000 description 1
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 1
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 1
- PURRNJBBXDDWLX-ZDLURKLDSA-N Ser-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N)O PURRNJBBXDDWLX-ZDLURKLDSA-N 0.000 description 1
- ILVGMCVCQBJPSH-WDSKDSINSA-N Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CO ILVGMCVCQBJPSH-WDSKDSINSA-N 0.000 description 1
- MFQMZDPAZRZAPV-NAKRPEOUSA-N Ser-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CO)N MFQMZDPAZRZAPV-NAKRPEOUSA-N 0.000 description 1
- ODRUTDLAONAVDV-IHRRRGAJSA-N Ser-Val-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ODRUTDLAONAVDV-IHRRRGAJSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 241000906446 Theraps Species 0.000 description 1
- CUTPSEKWUPZFLV-WISUUJSJSA-N Thr-Cys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CS)C(O)=O CUTPSEKWUPZFLV-WISUUJSJSA-N 0.000 description 1
- QQWNRERCGGZOKG-WEDXCCLWSA-N Thr-Gly-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O QQWNRERCGGZOKG-WEDXCCLWSA-N 0.000 description 1
- WXVIGTAUZBUDPZ-DTLFHODZSA-N Thr-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 WXVIGTAUZBUDPZ-DTLFHODZSA-N 0.000 description 1
- CRZNCABIJLRFKZ-IUKAMOBKSA-N Thr-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N CRZNCABIJLRFKZ-IUKAMOBKSA-N 0.000 description 1
- RFKVQLIXNVEOMB-WEDXCCLWSA-N Thr-Leu-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N)O RFKVQLIXNVEOMB-WEDXCCLWSA-N 0.000 description 1
- MEJHFIOYJHTWMK-VOAKCMCISA-N Thr-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)O MEJHFIOYJHTWMK-VOAKCMCISA-N 0.000 description 1
- MECLEFZMPPOEAC-VOAKCMCISA-N Thr-Leu-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MECLEFZMPPOEAC-VOAKCMCISA-N 0.000 description 1
- QJIODPFLAASXJC-JHYOHUSXSA-N Thr-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O QJIODPFLAASXJC-JHYOHUSXSA-N 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- GRQCSEWEPIHLBI-JQWIXIFHSA-N Trp-Asn Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O)=CNC2=C1 GRQCSEWEPIHLBI-JQWIXIFHSA-N 0.000 description 1
- WMBFONUKQXGLMU-WDSOQIARSA-N Trp-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N WMBFONUKQXGLMU-WDSOQIARSA-N 0.000 description 1
- NLMXVDDEQFKQQU-CFMVVWHZSA-N Tyr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NLMXVDDEQFKQQU-CFMVVWHZSA-N 0.000 description 1
- FXYOYUMPUJONGW-FHWLQOOXSA-N Tyr-Gln-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 FXYOYUMPUJONGW-FHWLQOOXSA-N 0.000 description 1
- WPXKRJVHBXYLDT-JUKXBJQTSA-N Tyr-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CC=C(C=C2)O)N WPXKRJVHBXYLDT-JUKXBJQTSA-N 0.000 description 1
- USYGMBIIUDLYHJ-GVARAGBVSA-N Tyr-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 USYGMBIIUDLYHJ-GVARAGBVSA-N 0.000 description 1
- CGWAPUBOXJWXMS-HOTGVXAUSA-N Tyr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 CGWAPUBOXJWXMS-HOTGVXAUSA-N 0.000 description 1
- SCZJKZLFSSPJDP-ACRUOGEOSA-N Tyr-Phe-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O SCZJKZLFSSPJDP-ACRUOGEOSA-N 0.000 description 1
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 1
- WQOHKVRQDLNDIL-YJRXYDGGSA-N Tyr-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O WQOHKVRQDLNDIL-YJRXYDGGSA-N 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- FTKXYXACXYOHND-XUXIUFHCSA-N Val-Ile-Leu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O FTKXYXACXYOHND-XUXIUFHCSA-N 0.000 description 1
- APQIVBCUIUDSMB-OSUNSFLBSA-N Val-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N APQIVBCUIUDSMB-OSUNSFLBSA-N 0.000 description 1
- DAVNYIUELQBTAP-XUXIUFHCSA-N Val-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N DAVNYIUELQBTAP-XUXIUFHCSA-N 0.000 description 1
- JKHXYJKMNSSFFL-IUCAKERBSA-N Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN JKHXYJKMNSSFFL-IUCAKERBSA-N 0.000 description 1
- WBAJDGWKRIHOAC-GVXVVHGQSA-N Val-Lys-Gln Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O WBAJDGWKRIHOAC-GVXVVHGQSA-N 0.000 description 1
- MJFSRZZJQWZHFQ-SRVKXCTJSA-N Val-Met-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)O)N MJFSRZZJQWZHFQ-SRVKXCTJSA-N 0.000 description 1
- LJSZPMSUYKKKCP-UBHSHLNASA-N Val-Phe-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 LJSZPMSUYKKKCP-UBHSHLNASA-N 0.000 description 1
- FMQGYTMERWBMSI-HJWJTTGWSA-N Val-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N FMQGYTMERWBMSI-HJWJTTGWSA-N 0.000 description 1
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- OFTXTCGQJXTNQS-XGEHTFHBSA-N Val-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N)O OFTXTCGQJXTNQS-XGEHTFHBSA-N 0.000 description 1
- JAIZPWVHPQRYOU-ZJDVBMNYSA-N Val-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O JAIZPWVHPQRYOU-ZJDVBMNYSA-N 0.000 description 1
- VEYJKJORLPYVLO-RYUDHWBXSA-N Val-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 VEYJKJORLPYVLO-RYUDHWBXSA-N 0.000 description 1
- PGBMPFKFKXYROZ-UFYCRDLUSA-N Val-Tyr-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N PGBMPFKFKXYROZ-UFYCRDLUSA-N 0.000 description 1
- JVGDAEKKZKKZFO-RCWTZXSCSA-N Val-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N)O JVGDAEKKZKKZFO-RCWTZXSCSA-N 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical class C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 230000001800 adrenalinergic effect Effects 0.000 description 1
- 239000000464 adrenergic agent Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- HMFHBZSHGGEWLO-LECHCGJUSA-N alpha-D-xylofuranose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@H]1O HMFHBZSHGGEWLO-LECHCGJUSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000003816 antisense DNA Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 208000004668 avian leukosis Diseases 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 108010069495 cysteinyltyrosine Proteins 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000009762 endothelial cell differentiation Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- IFJBPERYMVGYFG-PMACEKPBSA-N ethyl (2s)-2-[[(2s)-2-acetamido-3-[4-[bis(2-chloroethyl)amino]phenyl]propanoyl]amino]-4-methylsulfanylbutanoate Chemical compound CCOC(=O)[C@H](CCSC)NC(=O)[C@@H](NC(C)=O)CC1=CC=C(N(CCCl)CCCl)C=C1 IFJBPERYMVGYFG-PMACEKPBSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- KZNQNBZMBZJQJO-YFKPBYRVSA-N glyclproline Chemical compound NCC(=O)N1CCC[C@H]1C(O)=O KZNQNBZMBZJQJO-YFKPBYRVSA-N 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 239000006451 grace's insect medium Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 108091006093 heterotrimeric G proteins Proteins 0.000 description 1
- 102000034345 heterotrimeric G proteins Human genes 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 210000000003 hoof Anatomy 0.000 description 1
- 102000046768 human CCL2 Human genes 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 1
- 108010027338 isoleucylcysteine Proteins 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- DVCSNHXRZUVYAM-BQBZGAKWSA-N leu-asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O DVCSNHXRZUVYAM-BQBZGAKWSA-N 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 108010091798 leucylleucine Proteins 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000003574 melanophore Anatomy 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003551 muscarinic effect Effects 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 239000003176 neuroleptic agent Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000030788 protein refolding Effects 0.000 description 1
- 208000009305 pseudorabies Diseases 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- RYVMUASDIZQXAA-UHFFFAOYSA-N pyranoside Natural products O1C2(OCC(C)C(OC3C(C(O)C(O)C(CO)O3)O)C2)C(C)C(C2(CCC3C4(C)CC5O)C)C1CC2C3CC=C4CC5OC(C(C1O)O)OC(CO)C1OC(C1OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OCC(O)C(O)C1O RYVMUASDIZQXAA-UHFFFAOYSA-N 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000019254 respiratory burst Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002265 sensory receptor cell Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000007390 skin biopsy Methods 0.000 description 1
- 101150038846 slc46a1 gene Proteins 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- SRVJKTDHMYAMHA-WUXMJOGZSA-N thioacetazone Chemical compound CC(=O)NC1=CC=C(\C=N\NC(N)=S)C=C1 SRVJKTDHMYAMHA-WUXMJOGZSA-N 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 230000036964 tight binding Effects 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
-
- 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
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
-
- 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
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- 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
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Human G-protein chemokine receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the G-protein chemokine receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the G-protein chemokine receptor nucleic acid sequences and detecting a level of the soluble form of the receptors in a sample derived from a host.
Description
fi~JiN G-PROTEIN CgB~SOICIlQS RFCBPTOR BaiGHRlO
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and, polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human 7-transmembrane receptor which has been putatively identified as a chemokiae receptor, sometimes hereinafter referred to as "G-Protein Chemokine Receptor" or "HD~iRlO". The imrention also relates to inhibiting the action of such polypeptides.
It is well established that many medically significant biological processes are mediated by proteins participating in signa7~ transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 11991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG
proteins . Some examcples of these proteins include the GPC
receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., PNA.S, 84:46-50 (1987); Kobilka, B.K., et al., Science. 238:650-656 11987); Bunzow, J.R., et al., Nature, 336:783-787 (ig88)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein -la-kinase A and protein kinase C (Simon, M.I., et al., Science, 252:802-8 X1991)).
For example, in one form of signal transduction, the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. 8azyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone binding. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself, returns-the.G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intexinediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The ~mbrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane do~ai.ns. The domains are believed to represent traasmembrane a-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a pride range of biologically active receptors , such as hormone , viral, growth factor and neuroreceptors. ,.
G-protein coupled receptors have been characterized as including these seven consezved hydrophobic stretches of about 20 -to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include calcitonin, adrenergic, eadothelin; cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1 receptor and rhodopsins, odorant, cytanegalovirus receptors, etc.
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and, polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human 7-transmembrane receptor which has been putatively identified as a chemokiae receptor, sometimes hereinafter referred to as "G-Protein Chemokine Receptor" or "HD~iRlO". The imrention also relates to inhibiting the action of such polypeptides.
It is well established that many medically significant biological processes are mediated by proteins participating in signa7~ transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 11991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG
proteins . Some examcples of these proteins include the GPC
receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., PNA.S, 84:46-50 (1987); Kobilka, B.K., et al., Science. 238:650-656 11987); Bunzow, J.R., et al., Nature, 336:783-787 (ig88)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein -la-kinase A and protein kinase C (Simon, M.I., et al., Science, 252:802-8 X1991)).
For example, in one form of signal transduction, the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. 8azyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone binding. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself, returns-the.G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intexinediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The ~mbrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane do~ai.ns. The domains are believed to represent traasmembrane a-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a pride range of biologically active receptors , such as hormone , viral, growth factor and neuroreceptors. ,.
G-protein coupled receptors have been characterized as including these seven consezved hydrophobic stretches of about 20 -to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include calcitonin, adrenergic, eadothelin; cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1 receptor and rhodopsins, odorant, cytanegalovirus receptors, etc.
G-protein coupled receptors can be intrace~lularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion criannels and transporters (see, Johnson et al., Bndoc., Rev., 10:317-331 (1989)). Different G-protein a-subuaits preferentially stimulate particular effectors to modulate various biological functions in a cell.
Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling. of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.
Chemokines, also referred to as intercriae cytokines, are a subfamily of structurally and functionally related cytokines . These molecules are 8-10 kd in size . In general, chemokines exhibit 20~c to 75~ homology at the amino acid level and are characterized by four conserved cysteine~
residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta.
In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the "C-g-C"
subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the "C-C" subfamily. Thus far, at least nine different members of this family have been identified is humans.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblaets. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histaiaine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of coatplement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflam~aation, certain chemokines have bees shown to exhibit other activities . For examcple, macrophage inflanrcc~aatory protein 1 tMIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4 ) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells .
In light of the diverse biological activities, it is not surprising that che~nokiaes have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
In accordance with one aspect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present inveatioa'are of human origin.
In accordance with aaother~ aspect of the present invention, there are. provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as aatisense- analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect of the present imreation, there are provided processes for producing such receptor polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences encoding the receptor polypeptides of the present invention, under conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.
In accordance with yet a further aspect of the present invention, there are provided antib.~dies against such receptor polypeptides.
In accordance with another aspect of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of the receptor polypeptides of the present invention.
In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in stinn~lating haematopoiesis, around healing, coagulation, angiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-imaname diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.
In accordance with another aspect of the present invention there is provided a method of administering the receptor polypeptides of the present invention via gene therapy to treat conditions related to underexpression of the polypeptides or underexpression of a ligand for the receptor polypeptide_ In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and Ig$-m;ediated allergic reactions; prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosinophilic syndrome.
In accordance with yet another aspect oft. the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.
Tn accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the~present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding such polypepti.des, ~ for .in' vitro purposes related. ' to scientific research, synthesis of DNA and manufacture of DNA vectors.-These and other aspects of the present invention should be apparent to those skilled~in the art from the teachings herein.-The following drawings acre illustrative of embodiments of the invention and are not meant to limit the scope.of the invention as encompassed by the_claims.
Figure 1 shows the cDNA sequence and the. corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention. the tandard one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequ~ncer (~..pplied Biosystems, Inc.).
Figure 2 illustrates an amino acid alignment of the G-protein chemokine receptor~of the present invention and the human MCP-I receptor.
In accordance with an aspect of the preser_t invention, there is provided an isoxated nucleic acid (polyizucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID N0:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 97183 with the Rmerican Type Culture Collection, 12301 Parklawn Drive, Rockv?11e, Mary?and, 20852, United States of America, on June ~, 1995.
The polynucleot~~.de of this invention was discovered in a cDNA library derived from human monocytes. It is stiucturallv related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 352 amino acid residues. The protein exhibits the highest degree of homology to a human MCP-1 receptor with 70.1 %
identity and 82.9 % similarity over a 347 amino acid stretch.
The polynucleotide of the present invention may be is the form of RNA or in the forca of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DN~.1 may. be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (S$Q ID
NO:1) or that of the .deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the-same mature polypeptide as the DNA of Figure 1 (S8Q ID N0:1)~
or the deposited cDl~.
.The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by~the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for- the mature palypeptide and additional-coding sequence such as a transmembraae (T1~1')- or intro-cellular domain; the coding sequence for the . mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence far the mature polypeptide. ' Thus, the term "polynucleotide encoding a polypeptide"
encooapasses a polynucleotide which includes only codir~g sequence for the polypeptide as well as-.a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the herei.nabove described polyaucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amigo acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. T~.
variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (S$Q ID N0:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as .variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SBQ~ID
NO:~) or the polypeptide encoded by the cDI~ of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the.polyaucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown is Figure 1 (S$Q ID-NO:~)_or of the coding sequence of the deposited clone. As known is the art, as allelic variant is as altezaate form of a polyaucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide_ ' The polyaucieotides'may also encode for a soluble form of the G-protein chemokine receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved froia the TM and intracellular domain of the full-leagth poiypeptide of the present invention.
The polynucleotides of the present~invention may also have the coding sequence fused~in frame to a marker sequence which allows for purification of the polypeptide of the present imrention_ The marker sequence may be a hexa-histidine tag supplied by a pQB-9 vector to provide far-purification of the mature polypeptide fused to the marker is the case of a~bacterial host, or, for example, the marker sequence may be a hemagglutinin~(HA) tag when'a-mammalian _g_ host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:76: (1984)y.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (axons).
Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA~
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases . The probe may also be used to identify a cD~~iA
clone corresponding to a full length transcript and a genomic ;
clone or clones that contain the complete gene including regulatory and pranotor regions, axons, and introns. An example of a screen coomprises isolating the coding region of the gene by using the laio~rn DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, ;.geaomic DATA or mR~ to determine which members of the library the probe hybridizes to.
The present invention further relates to polynucleotides which hybridize to the hereiaabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The-present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereiaabove-described polynucleotides. As herein used, the terra "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides _9-which hybridize to the herei.na.bave described polynucleotidE
in a preferred embodiment encode polypeptides which either retain substantially the same biological function yr activity as the mature polypeptide encoded by the cDl~s of Figure 1 (SBQ ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and awre preferaGbly at least 50 bases which hybridize to a polynucleotide'of the present imrention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SBQ ID I~IO:1, for ~ exan~le, for recovery of the polyaucleotide or as a diagnostic probe or as a PQ2 primer.
Thus, the present invention is directed to polynucleotides having at~ least a 70~ identity, preferably at Least 90~ and more preferably at least a 95~ identity to a polynucleotide which encodes the polypeptide of SBQ ID N0:2 as well as fraga~eats thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polyaucleotides.
The deposits) referred to herein will be maintained under the terms of, the Budapest Treaty on the International Recognition of the Deposit of Micro-organistas for purposes of Patent Procedure. These deposits ajre provided merely as convenience to those of skill ia-the art and are not an adatission that a deposit is required.
The segueace of the polynucleotides contained in the deposited materials, as well. as the axaino acid sequence of the polypeptides encoded thereby, are controlling in the event of nay conflict.
with~any description of~sequences herein. A license utay be required to make, or sell the deposited materials, and no such license is hereby granted..
_1Q_ The present invention further relates to a G-protein chemokine receptor polypeptide which has the deduced amino acid sequence of Figure 1 (SBQ ID N0:2) or which has the amino acid sequence encoded by the deposited'cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The terns ~fragment," "derivative" and ~anaiog~ when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e_ functions as a G-protein chemokine receptor, or retains the ability to bind the ligand or the receptor even though_the polypeptide does not function as a G-protein chemokine receptor, for example, a soluble form of the receptor. An analog includes a proprotein~which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polyQeptide of Figure 1 (SNQ ID N0:2) or that encoded by the deposited cDI~ may be (i) one in which one -or more of the am;.ao acid residues are substituted with a conserved or non-consezved amino acid residue (preferably a conserved amino acid residue)~and such substituted amino acid residue may or may not be one encoded by the genetic code, or <ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a comcpound to increase the half-life of the- polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide or (v) one in which a fragment of tie polypeptide is soluble, i.e. not membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives and analogs are deeated to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The polypeptides of the present invention include the polypeptide of SBQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably a 70% identity) to the polypeptide of SBQ ID N0:2 and more preferably a 90% similarity (more .
preferably a 90% identity) to the polypeptide of SBQ ID N0:2 and still more preferably a 95% similarity (still more preferably a 90% identity) to the polypeptide of S$Q ID N0:2 and to portions of such polypeptide with such portion of the poiypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The tezm "geese" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons)_ The term "isolated° means that the material is removed from its original environment (e. g., the natural environment if it is naturally occurring). Por example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from s~aae or all of the coexisting ~aaterials is the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a co~o~position, and still be isolated in that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SSQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at least 70% identity) to the polypeptide of S8Q ID N0:2 and more preferably at least 90%
similarity (more preferably at least 90% identity) to the polypeptide of S$Q ID N0:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SBQ ID N0:2 gad also iaciude portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amigo acids.
~ ~o~ ~ the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second po3ypeptide.
Frac~nents or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides: Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The present invention also relates to vectors which include polyaucleotides of the present invention, host cells which are genetically engineered with vectors 'of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating.
promoters , selecting transfo~mants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant tec~iques. Thus, for example, the polyaucleotide may be included in nay one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of Sv40; bacterial plasmids; phage DNA;
bacuZovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral D1~ such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicabie and viable in the host.
The appropriate DNA seguence may be inserted into~the vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction endonuclease sites) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequences) (promoter) to direct mRNA synthesis. As representative examples of such pramoters, there may be mentioned: LTR or SV4Q promoter, the E. coli. lac or t~, the phage lambda P~
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their vizuses.
The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in B. coli.
The vector containing the appropriate DID sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to traasforar an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, StreDtonHrces, SalaaonelZa tvnhimurium; fungal cells, such as yeast; insect cells such as Drosonhila and cetera animal cells such as CEO, COS or Hooves melanoma; adenovirus;
plant cells, etc. The selection of an appropriate host is deemed to.be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs co~ac~prise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably _Z~_ linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are comseercially available. The following vectors are provided by way of example. Bactei_al: pQ$70, pQB60, pQ$-9 (Qiagen), pbs, pDlO, phagescript, psiXi74; pbluescript SIC, pbsks, pl~8A, pNXl6a, pN818A, pNH46A (Stratagene); ptrc99a, pKR223-3, pKR233-3, pDR540, pRITS (Pharmacia). Bukaryotic: pWLN80, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3; pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable aad viable in the host.
Promoter regions can be selected from aay desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL aad trp.
Bukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, aad mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level. of ordinary skill in the art.
In a further embodiment, the_present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaxyotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DSAB-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (198x)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from ' the Did constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).
Transcription -of the DIZA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Bnhancers are cis-acting elements of DID,, usually about from 10 to 300 by that act on apromoter to increase its~transcription.
Examples including the SV40 eahancer on the late side of the replication origin by 100 to 270, a cytoa2egalovirus early promoter eahancer, the polyoma exl~ancer on the late side of the. replication origin, and adeziovirus enbancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae T1ZP3 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and ter~aination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular meditaa. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization~or simplified purification of expressed . recombinant product.
Useful expression vectors for bacterial use are constxucted by inserting a structural DNA seguence encoding a desired protein together With suitable translation initiation and tezmination signals in operable reading phase with a functional promoter. The vector will.comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts' for transformation include B. coli, Bacillus subtilis, Salmonella tvahimurium and various species within the ~ genera Pseudomoaas, Streptomyces, and .Staphylococcus, although others may also be employed as a~
matter of choice. ~ ' As a representative but noalimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin~of replication derived from comsterczally available plasmids cocaprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such c~ercial vectors include, for eacample, pKK223-3 (Pbar<nacia Fine Chemicals, Uppsala, Sweden) and G8M1 (Promega Biotec, Madison, WI, tTSA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e. g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, diszupted by physical or chemical means, and the resulting crude extract retained for further purification.
_18_ Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
various mammalian cell culture systems can also be employed to express recombinant protein. fixamples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C12?, 3T3, C80, Fieha and BHK cell lines. Mammalian expression vectors will coa~rise an origin of replication, a suitable promoter and eahancer, and also any necessary ribosa~ne binding sites, polyadeaylation site, splice donor and acceptor sites, transcriptional termination segueaces, and 5' flanking nontranscribed sequences. D1~1 sequences derived from the SV40 splice, and polyadeaylation sites may be used to provide the required nontranscribed genetic elements.
The G-protein che~xaokine receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial methionine amino acid residue.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.
The G-protein chemokine receptors of t3ie present im~ention may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide of the present invention .
In general, such screening procedures involve providing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, yeast, drosophila or E. Cola. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the G-protein chemokiae receptor. The expressed receptor is then contacted with a test compound to obse3cve binding, stimulation or inhibition of a functional response.
One such screening procedure involves the use of ~aelanophores which are traasfected to express the G-protein chemokine receptor of the present invention. Such a screening technique is described in PCT WO 92 / 01810 published February 6, 1992.
Thus, for example, such assay may be employed for screening for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.
The screen may be employed for determining a compound which activates the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.
Other screening techniques include the use of cells which express the G-protein chemokine receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (October 1989). For example, compounds may be contacted with a cell which expresses the receptor polypeptide of the present imrention and a second messenger response, e.g.-signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.
Another such screening technique involves introducing RtsA encoding the G-protein chemokiae receptor into 8enopus oocytes to transiently express the receptor.. The receptor oocytes may then be contacted with the receptor ligand and a cooa~pound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for caa~pounds which are thought to inhibit activation of the receptor.
Another screening technique involves expressing the G-protein chemokine receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embzyonic kidney cells, etc. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.
Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting .a eukaryotic cell with DNA encoding the G-protein chemokine receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the coarpound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
An antibody may antagonize a G-protein chemokine receptor of the present invention, or in so~e cases an oligopeptide, which bind to the G-protein chemokiae receptor but does not elicit a second messenger response such that the activity of the G-protein chemokine receptors is prevented.
Antibodies include anti-idiotypic antibodies which recognize unique determinants generally associated with the antigen-binding site of as antibody. Potential antagonist compounds also include proteins which are closely related- to the Iigaad of the G-protein chemokine receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein chemokine receptor elicit no response.
An aatisease construct prepared through the use of aatisense technology, may be used to control gene expression through triple-helix formation or aatisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DTiA or RIB. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an aatisease RNA oligoaucleotide of frost about 1f to 40 base pairs in length. A DNA oligonucleotide is designed to be coanplemeatary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988):
and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of G-protein chemokine receptor. The antisense RNA oligonucleotide hybridizes to the ~aaRNA in vivo and blocks translation of mRNA
molecules into G-protein coupled receptor (antisense - Okano, J. Neurochem.. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Baton, FI: (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA
may be expressed ~En vivo to inhibit production of G-protein chemokine receptor.
A small molecule which binds to the G-protein chemokine receptor, making it inaccessible to ligands such that nozmal biological activity is prevented, for example small peptides or peptide-like molecules, may also be used to inhibit activation of the receptor polypeptide of the present invention.
A soluble f ozln of the G-protein chemokiae receptor, a . g .
a fragment of the receptors, may be used to inhibit activation of the receptor by binding to the ligand to a polypeptide of the present invention and preventing the ligand from interacting with membrane bound G-protein chemokiae receptors.
The com~pouads which bind to and activate the G-protein chempkine receptors of the present invention may be employed to stite haematopoiesis, wound healing, coagulation, aagiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-im~awae diseases, parasitic affections, psoriasis, and to stimulate growth factor activity.
The compounds which bind to and inhibit the G-protein chemokine receptors of the present invention may be employed to treat allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and Ig$-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis shock and hyper-eosinophilic syndrome.
The compounds may be employed in combination with a suitable pharmaceutical carrier. Such'compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should ~ suit the mode of administration.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of. the pharmaceutical compositions of the invention. Associated with such containers) can be a notice in the fozm prescribed by a governmental agency regulating the manufacture, use or sale. of pharmaceuticals or biological products, which not~.ce reflects approval by the agency of _asanufacture, use or sale for human administration_ In additio'~n, the co~apouads. of the present invention may be employed in conjunction with other therapeutic coxapounds.
The pharmaceutical ,cocapositions may be ac~ainistered in a convenient manner such as by the topical, intravenous, iatraperitoaeal, iatramuseular, subcutaneous, iatraaasal or 3atradermal (applicable) routes. The pharmaceutical compositions are administered in as amount which is effective for treating and/or prophylaxis of the specific indication.
In general, the phaanaceutical compositions will be administered in an amount of at least about ZO ~eg/kg body weight and in nmost cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day.
In most cases, the dosage is from about 10 ,ug/kg to about 1 mg/kg body weight daily, taking into account the routes of adnttnistratioa, symptoms, etc., The G-protein chemokine receptor polypeptides and antagonists or agonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as ~gene therapy.~
Thus, for example, cells from a patient may be engineered with a polyaucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA
encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide is vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the preseat invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
R,etraviruses frown which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloaey Marine Leukemia Vizus, spleen necrosis vizus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis vines, gibbon age leukemia virus, human immunodeficiency virus, adenovirus;
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
In one embodiment, the retroviral plasmid vector is derived from Moloney Marine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (HIV) promoter described in Miller, et al., $~otechnioues, Vol. 7, No. 9, 980-990 (1989) , or any other promoter (e. g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a. suitable promoter. Suitable, prou~ot~rs which may be employed include, but are not limited to, adeaoviral promoters, such as the adenovirai major late proafoter; or hetorologous promoters, such as the cytomegalovirus (City) propaoter; the respiratory syncytial virus (RSV) praanoter; inducibie~promoters, such as the MMT promoter, the metallothionein proiaoter; heat. shock promoters; the alhumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thyad.dine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described).; the ~B-actin promoter; and human growth hormone promoters . The proutoter also ~ may be the native proamoter which controls the genes encoding the polypeptides.
The retroviral plas=aid vector is employed to transduce packaging cell lines to foza~ producer cell lines. Bx,amples of packaging cells which may be transfected include, but are not limited to, the PSSOl, PA337, ~-2, ~-AM, PA12, T19-14X, VT-39-I7-Fi2, s~CRB, ~C'RIP, GP+E-.86, GP+eavAml2, and DAN cell lines as described in Miller, Human Gene Theratw, vol. 1, pgs. 5-14 (1990) .
The vector may transduce the packaging cells through any means laiown in the art. Such means include, but are not limited to, electroporation; the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral 'plasmz.d vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequences) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either za vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequences) encoding the polypeptide. 8ukaryotic cells which may be transduced include, but are not limited to, embryoaic stem cells, embryonic carcinoma . cells, as well as hema_topo~:etic. stem . cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, aad bronchial epithelial cells.
The present invention also provides a method for determining whether a ligand not kaown to be capable of binding to a G-protein cheruokine receptor can bind to such receptor which comprises contacting a mammalian cell which expresses a, G-protein chemokine receptor-,with the ligand under conditions percaitting binding of ligands to . the G-protein chemokine receptor, detecting the presence of .a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein chemokine receptor.
The systems hereinabove described-for determining agonists and/or antagonists may also 'be employed for determining ligaads which bind to the receptor.
This invention also provides a method of detecting expression of a G-protein chemokine receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA coding for the receptor which comprises obtaining total mRHA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression of the receptor by the cell.
The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. . These related receptors .rnay be identif led by hortnology to a G-protei a chemokine receptor polypeptide of the present invention,-by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the chemokine receptor.polypeptides of the present invention.
Fragments of the genes may, be ~ used as a hybridization probe for a cDi~.1 library to isolate other genes which have a high sequence similarity to the genes of- the present invention, or which have similar biological activity. Probes of this type are at least 20 bases, preferably at least. 3.0 bases and angst preferably at least 50 bases or more. The probe may also be used to identify a cDI~1 clone corresponding to a full length'transcript and a genomic clone or clones that contain the caa~plete gene of the present invention including .regulatory and promoter regions, exons and introas .
An example of a screen of tb.is type comprises isolating the coding region of the gene by using the known DNA sequence to syathesize~ an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the. genes of the present invention are used to screen a library of human cDNA, genosaic DNA or mRNA to deter~ni.ae which members of the library the probe hybridizes to.
The present invention also contecuplates, the use of the genes of the present invention as a diagnostic, for example, score diseases result from inherited defective genes. .These genes can be detected by cooaparing the sequences of the defective gene with that of a normal one. Subsequently, one can verify that a "mutant" gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expression in a functional assay system (e. g., colorimetric assay, expression on MacConkey plates, c~rplementation experiments, in a receptor deficient strain of ~IC293 cells) as yet another means to verify or identify mutations. Once "mutant" genes have been identified, one can they screen population for carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techaigues. Nucleic acids used for diagnosis may be obtained froaa a patient' s cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material_ The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR
(Saiki, et al., N~.tu~, 324:163-166 1986) prior to analysis.
RNA or cDNA may also be used for the same purpose. As an example, PCR primers como~plimentazy to the nucleic acid of the instant invention can be used to identify and analyze mutations in the gene of the present invention. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to. radio labeled RNA of the invention or alternatively, radio labeled aatisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting te~peratures. Such a diagnostic would be particularly useful for prenatal or eves neonatal testing.
Sequence differences between the reference gene and "mutants" may be revealed by the direct DNA seqeiencing method. in addition, cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For exa~aa~ple, a sequence primer is used with double stranded PCR
product or a single stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radio labeled nucleotide or h:
an autoioaatic sequencing procedure With fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at specific locations may also be revealed by nucleus protection assays, such RNase and S1 protection or the chemical cleavage method (e.g. Cotton, et al. , PI~~~g. USA, 85:4397-4401 1985) .
In addition, sane diseases are a result of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be used as a reference to identify individuals expressing a decrease of functions associated with receptors of this type.
The present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the G-proem chemokiae receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well kaoarn to those of skill in the art and include radioimmvaoassays, competitive-binding assays, Western blot analysis and preferably as BLISA assay.
An BLISA assay initially coa~rises preparing an antibody specific to antigens of the G-protein chemokine receptor polypeptides, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzycae. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any G-protein chemokine receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody'is washed ou~ with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to G-protein chemokine receptor proteins. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of G-protein chec~okine receptor proteins present in a given volume of patient sample when cort~ared against a standard curve.
The seguences of the present invention are also valuable for chroancso~ne identification. The sequence is specifically targeted to and can hybridize with a particular location on as individual human chromoso~ane.. Moaceover, there is a current need for identifying particular sites on the chromosome . Few chro~some marking reagents based on actual sequence data (repeat polymo~cphisms) are presently available for marking chro~nosoma,,l location. The mapping of DNAs to chromosomes according to the present invention is an important f first step in corzelating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from' the cDNA.
Computer analysis of the cDNA is used to rapidly select primers that do not span more than one axon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell -3~-hybrids containing iadividtial human chro~awsomes. Oniy.those hybrids containing the human gene corresponding to the primer will yield an amplified fragaaent.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large geaomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in s~ttu hybridization, prescreening With labeled flow-sorted chromosomes and preselection by hybridization to construct chromaosome specific-cDl~ libraries.
Fluorescence ~fn situ hybridization (FISH) of a cDN~I
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York ( 1988 ) .
Once a sequence has been mapped to a precise chromosomal location, the physical position, of the sequence on the chroaaosome can be correlated with genetic map data. Such data are found, for example, in v. McRusick, Meadelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have bees mapped to the same chromosomal region are they identified through linkage analysis (coinheritarice of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals . If a mutation is observed in sa~ae or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes z megabase mapping resolution and one gene per 20 kb) .
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an im~nuaogen to produce antibodies thereto. These 'antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be~
obtained by direct injection of the polypeptides into an animal or by ad~ainisterinc the polvpeptides to an animal, preferably a nonhuman . The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment,of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human E-cell hybridama. technique (Rozbor et al : , 1983 , Imaaunology Today 4 : 72 ) , and the BBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies tll.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, tram~Anic mice may be used to express humanized antibodies to imvanunogenic polypeptide products of this imrention.
The present i~m~ention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate understanding of the following examples certain freguently occurring methods and/or terms will be described.
"Plasmids~ are designated by a lower case p preceded aad/or followed by capital letters and/or numbers. The starting plasmids herein are either co~mmnercially available, publicly available on an unrestricted. basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~cg of plasmid or DNA
fragment is used with about 2 units,of enzyme in about 20 ~cl of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~cg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzyr~aes are specified by the manufacturer . Incubation times of about 1 hour at 3 7~' C are ordinarily used, but may vary in accordance with the supplier s instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide geI described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be che~aically synthesized. Such synthetic oligonucieotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP is the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomaplished using known buffers and conditions with 10 units to T4 DNA lipase ("lipase") per 0.5 ycg of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Bb, A., Virology,.52:456-457 (1973).
Exa~le 1 Bacterial Bxflression and Purification of HDGNR10 The DNA sequence encoding for 5Z~R10, ATCC # _ is initially amplified using PCR oligonucleotide primers corresponding to theca 5' and sequences of the processed ~RlO protein (minus the signal peptide sequence) and the vector sequences 3' to the F~GNR10 geese. Additional r nucleotides corresponding to FmGYRIO were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence S' CGGAATTCCTCCATGGATTATCAAGTGTCA 3' contains an BcoRI restriction enzyme site followed by 18 nucleotides of H~N'RIO coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5~' CGGAAGCTTCGTCACAAGCCCACAGATAT 3' contains complementary sequences to ~ HindIII site and-is followed by z8 nucleotides of F~GNR10 coding sequence. The restriction enzyme sites correspond to the restr~.ctioa enzyme sites on the bacterial expression vector pQE-9 .(Qiagen, inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQB-9 encodes antibiotic resistance (A~up') , a bacterial origin of replication (ori) , as IPTG
regulatable promoter operator (P/0), a ribosome~binding site (RBS), a 6-His tag_and restriction enzyme sites. pQE-9 Gras then digested- with EcoRI and HindIII. The amplified t"
sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding.for the histidine tag and the RBS. The ligation mixture was then used to transforiu E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described zn Saiabrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (3989). M15/rep4 contains paultiple copies of~the plasmid pRFP4, which expresses the lacl repressor and also confers kanaa~ycin resistance (Xan°) .
Tzansfortaants are identified by their ability to grow on L8 plates and an~picillin/kanamycin resistant colonies were selected. Plasaaid , DNA was isolated and confizzaed by restriction analysis. - Clones containing the desired constnzcts were grown overnight (0/N) in liquid culture in Ia8 media supple~oaented with both Amp (Z00 ug/zal) and Kan (25 ug/ZN.). The O/N culture is used to inoculate a large culture at a ratio of 1.100 to- 1:250. The cells were grown tq an optical density 600 (0.D.°°°) of between 0.4 and 0.6.
IPTG
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing. the ?/0 leading to increased gene expression. Cells were grown an extra 3 to 4 hours .
-36=
Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCi. After clarification, solubilized HDGNR10 was purified from this solution by chromatography on a Nickel-Chelate~
column under conditions that allow for tight binding by groteins containing.the 6-His tag. Hochuli, E. et al., J.
Chromatography x11:17?-I84 (198x). HDGNR10 was eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine KC1, lO.OmM.sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione ~ (oxidized) . After incubation iri this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.
example 2 The expression .of plasrnid, HDGNR10 Iii is :derived front a vector pcDNAI/AmpM(Invitrogen) containing: 1) ~SV40 origin of replication, 2) ampicillin resistance gene, 3) E_coli.
replication origin, a) CMV promoter followed by a polylinker region, a SVaO intron and polyadenylation site. A DNA
fragment encoding the entire HDGNR10 precursor and a HA tag fused=in frame to its 3~ end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
.Niman, R. I~eighten,. A Cherenson, M. Connolly; and R. Lerner, 198x, Cell 37, 7~7) . The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction, strategy is described as follows:
The DNA sequence encoding for HDGNR10, ATCC No. 97L83, was constructed by PCFt using two primers: the S~ primer 5' GTCC
AAGCTTGCCACCATGGATTATCAAGTGTCA 3' and contains a HindIIT sit~-followed by 18 nucleotides of HDGNR10 coding sequence starting from the initiation colon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTt:TGGGACGTC~TATGGGTAGCACA.AGCCCACAGATATTTC
3' contains complementary ' sequences to an XhoI site, translation stop colon, HA tag and the last l8 nucleotides of the F~GNR10 coding sequence (not including the stop.codon).
Therefore, the PCR product contains a HindIII site F~GNR10 coding sequence followed by. HA tag fused in frame, a translation termination stop colon next to the HA tag, and an XhoI.site. The PCR amplified DNA fragment and the vector, pcDl~tAI/Amp, were digested with HindIII and Xhol restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning' Systems, 11099 North Torrey Pines Road, La Jolla, CA 92437) the transformed culture was - plated on atapicillin media plates and resistant ~ colonies were selected. Plasraid DNA was isolated from transfozmants and PYam~ned by restriction analysis for the presence of the correct fragment. For expression of the recoiabinant ~GNR~.O, COS cells were transfected 'with the expression vector by DBAE-DEXTRAN
method. (J. Sambrook, F. Pritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory press, (3989)). The expression of the HDGNR10 HA protein was detected by radiolabelling and iman:noprecipitation method.
(8. Harlow, D. Lane, Antibodies:. A Laboratory Manual, Cold Spring Harbor Laboratory Press, (i988)). Cells were labelled for 8 hours with '~S-cysteine two days past transfection.
Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 :nM NaCI, 1~ NP-40, 0.1~ SDS, i~~
NP-40, 0.5$ DOC, 50mM Tris, pH 7.5). (Wilson, I. et al " Id.
37:767 (1984)x. Both cell lysate and culture media were precipitated with a EA specific monoclonal antibody:
Proteins precipitated were analyzed on 15~c SDS-PAGE gels.
F.~s.~~.~tl a 3 n th v' g~grP~sion svstem The DNA, sequence encoding the full length HDGNR10 protein, ATCC No. 97183, 'was amplified using PCR
oligonucleotide primers corresponding to the 5' .and 3' sequences of the gene : ' The S' primer has the sequence 5' CGGGATCCCTCCATGGATTAT
CAAGTGTCA 3' and contains a BamHI restriction enzyme. site followed by 4 nucleotides resembling-anlefficient signal for the initiation of translation in eukaryotic cells (J. Mol.
Biol. 1987,-196, 9Q7-950, Kozak, M.), and just behind the first 18 nucleotides of the HDGNR10 gene (the initiation codon for translation is "ATG"). ' The 3' primer has the sequence 5' CGGGATCCCGCT
CACAAGCCCACAGATAT 3' and container the cleavage site for the restriction endonuclease BamHI and- 18nucleotides complementary- to the 3' non-translated sequence of the HDGNRIO gene. The amplified 'sequences were isolated from a I% agarose gel using a commercially available kit ( "Geneclean, " BIO 10I Inc . , La -Jolla, Ca . ) . The fragment was then digested with the endonuclease BarnFII -and purified as described above. This fragment is designated F2.
The vector .pRGl (modification. of pvL9~1 'vector, discussed below) is used for the expression of the HDGNR10 protein using the baculovirus expression system (for revie~r see: Summers, M.D, and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural- Experimental Statiori Bulletin No. 1555) .
This expression vector contains the stroizg polyhedrin promoter of the Autographs californica nuclear polyhedrosis virus (AcMNPV) -followed by the recognition sites for the restriction endonuclease BamFiI. The polyadenylation site of the simian virus (SV}40 is used for efficient polyadenylation. For an easy selection of recomb~~nant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed ' by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. aad Suaaners, M.D. , Virology, 170:31-39) . .
The plasmid was digested With the restriction enzyme BamHI and then dephosphorylated usiag calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1~ agarose gel as described above. This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA lipase. ls.coli HS101 cells were then transformed and bacteria identified that contained the plasmid (pBacBDGrTRIO ) with the HDC;NR10 gene using the eazyn~e BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.
~Cg of the plas-.nid pBac~GNRlO were co-transfected with i.0 ~cg of a caaamercially available liaearized baculovirus ("BaculoGold" baculovirus DNA°, Phazmingen, Saa Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
leg of BaculoGold'"' virus DNA and 5 ~g of the plasmid pBacBDGrTRIO were mixed in a sterile well of a microtiter plate containing 50 ~ul of serum free Grace's meditma (Z.ife Technologies Inc., Gaithersburg, MD). Afterwards 10 girl Lipofectin plus 90 ~Cl Grace's median were added, mixed and incubated for 15 miautes at room t~perature. Then the..
transfection mixture Was added drop wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 n~ tissue culture plate with 1 ml Grace' medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27°C. After S hours .the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf senun was added. The plate was put back into an incubator and cultivation continued at 27°C for four days.
After four days the supernatant was collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows as easy isolation of blue stained plaques. ~ (A
detailed description of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life_Technologies Inc., Gaithersburg, page 9-10) _ Four days after the serial dilution, the viruses were added to the cells, blue stained plaques were picked with the tip of an Eppendarf pipette. T3~e agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing~200 ~.l of Grace's medium.. The agar was removed by a brief centrifugation and the supernatant containing. the recombinant baculoviruses was used to infect Sf9 cells seeded in 35 moa dishes. Four days later the supernatants of these culture dishes were harvested and~then stored at 4°C.
Sf9 cells were grown in Grace's anedium~supplemented~with 10% heat=inactivated FBS_ The cells were infected with the recombinant baculovinxs V-~GNR10 at a multiplicity of infection tMOI) of 2. Six hours later the medium Was removed and replaced with SF900 II mediuui minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later S ~cCi of ~S-methionine and 5 ~.Ci 'sS cysteine (Amersham) were added. The cells were further incubated for i6 hours before they were harvested by centrifugation and the Zabelled proteins visualized by SDS-PAGE and autoradiography.
Exatrmle 4 - ExoressiQn via Gene Therap~r Fibroblasts are obtained from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10~ FBS, penicillin and streptomycin, is added.
This is then incubated at 37°C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayex is trypsinized and scaled into larger flasks.
pMV-7 (Rirsctmneier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloaey marine sarcoma virus, is digested with BcoRI and HiadII2 and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR prisoners which correspond to the 5' and 3' end sequences respectively: The 5' primer ccntains an $coRI sits and the 3' primer contains a HindIII site. $qual quantities of the Moloney murinE sarcoma virus linear backbone and the BcoRI and HindIII fragment are added together, in the presence of T4 DNA lipase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA3l~ or GP+aml2 packaging cells are grown in tissue culture' to confluent density in Dulbecco~s Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin arid streptomycin. The.MSV vector containing the ' gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (.the packaging cells are now referred to as producer cells).
Fresh ~aedia is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the_media from the producer cells. This media is removed and replaced with fresh med~,a. ~ If the titer of vixus is high, then virtually all fibroblasts will be infected and no~selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or hid.
The engineered fibroblasts are then infected into the ho$t, either alone or after~~having been grows to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
Numerous modifications and variations of the present invention are possible in light of the above~teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: HUMAN GENOME SCIENCES, INC. .
ROCKVILLE, MD 20850 UNITED STATES OF AMERICA
APPLICANT/INVENTOR: LI, Yi RUBEN, Steven M.
_, (ii) TITLE OF INVENTION: IiUMAN G-PROTEIN CHEMOKINE RECEPTOR HDGNRIO
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
(B) STREET: 1100 NEW YORK AVE., NW, SUITE 600 (C) CITY: WASHINGTON
(D) STATE: DC
(E) COUNTRY: USA
(F) ZIP: 20005 (v) COMPtTTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible - .
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,216,912 (B) FILING DATE: 06-JUN-1995 (C) CLASSIFICATIdN:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US95/07173 (8)- FILING DATE: 06-JUN-1995 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: STEFFE, ERIC K.
(B) REGISTRATION NUMBER: 36,688 (C) REFERENCE/DOCKET NUMBER: 1488.I15CA00 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 371-2600 (B)..TELEFAX: (202) 371-2540 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1419 base pairs (B) TYPE: nucleic acid (C). STRANDEDNESS: double ~ .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATDRE:
(A) NAME/FEEY: CDS
(B) LOCATION: 259._131!
(xi) SEQUE1~TCE DESCRIPTION: SEQ ID NO:1: _ GGAAGCTAGC AGCAAAGCTT CCCTTCACTA CGAAACTTCA TTGCTTGGCC CAAAAGAGAG z20 GCATTCATGG AGGGCAAC2A'AATACFsTTCT AGGACTTTAT AAAAGATCAG TTTTTATTTA 240 Met Asp Tyr 61~x Val Ser Ser Pro Ile Tyr Asp ATC AAT TAT TAT gCA TCG 6AG CCC ~'GC CCA AAA ATC AAT GTG AAG CAA . 339 Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Pro hys Ile Asn Val Lys~ Gln 15 20 ~ 25 IIe~Ala Ala'Arg Leu he~u Pro Pro heu Tyr Ser Leu Val Phe Ile Phe 30 35 - . 40 GGT TTT GTG GGC AAC ATG CTG GTC ATC CTC _AT~C CTG ATA AFiC TGC CAA ~ ' 435 Gly Phe Val Gly Asa Met Leu Val_ Ile ~.eu Ile Leu Ile Asa Cys Gln 45 54 ~ SS
AGG CTG GAG AGC ATG ACT GAC ATC TAC CTG CTC AAC CTG GCC.ATC TCT 9S3 Arg beu GIu Ser Met Thr Asg I1e Tyr Z.eu F.eu Asia Le3~ Ala Ile Ser 6f? 65 70 75 .
GAC CTG TTT TTC CTT CTT ACT GTC CCC TTC TGG ~GCT CAC 1'l~lT GCT GCC 53I
Asp Leu Phe Phe.heu l.eu- Thr VaI Pro Phe Trp Ala 8is Tyr Ala Ala '. 80 85 90 GCC CAG TGG GAC TTT GGA AAT ACA. 1$TG T'GT CAA CTC T~'G' ACA. GGG CTC ' 579' Ala Gln Trp Asp Phe Gly Asn Thr Met Cys GIr~ _ F~e~x Few TY~rr Gly heu Tyr Phe Ile Gly Phe Phe Ser Gly Tle Phe Phe IIe Ile Ixtz beu Thr lla ~~.s ~ 120 . . ~ .
ATe c~T AGG~TAC eT~ GCT ATC GTC C~~ GCT.GTG TTT GCS TTA AAA cce _. s7s ..
I2e Asp Arg Tyr Lew Rla Ile Val F~is ~ Ala ~i'af Phe F~a~ ~ ~eu ~.ys P~.a 12S ~ 134 . 135 AGG ACG GTC ACC TT'F GGG GTG GTG~ACA AGT GTG ATC ACT TG6 6TG GTG 723 Arg Thr Val Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp Pal Val Ala Va2 Phe Ala Sec heu Pro Gly Ile fle Phe Thr Arg Ser Gln Lys 16c~ lss ~ loo GAA~GGT CTT CAT TAC ACC TGC AGC TCT CAT TTT CCA TAC AGT CAG TAT 819 Glu Gly Leu His Tyr Thr Cys Ser Sex ~~is.Phe Pro Tyr Ser Gln Tyr z~7s . zeo 18s -q.s-CAA TTC TGG AAG AAT TTC CAG ACA TTA AF~G ATA GTC ATC TTG GGG CTG 867 Gln Phe Trp Lys Asn Phe Gln.Thr Leu Lys Ile Val Ile heu Gly Leu Val Leu Pro Leu leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys ACT"CTG CTT CGG TGT CGA AAT GAG AAG AAG AGG CAC AGG GCT GTG AGG 963 Thr Leu f~eu Arg Cys Arg Asn Glu r.ys Lys Arg His Arg~ Ala Val Arg CTT ATC TTC ACC ATC ATG ATT GTT TAT TTT CTC TTC TGG GCT CCC TAC ' 1011 Leu Ile Phe Thr Ire Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr .
24'O 245 250 Asn Ile Val Leu Leu Leu Asn Thr Phe Gln Glu Phe Phe~ Gly Leu Asn AGC AGG TTG
GAC CAA
GCT ATG
Asn Cys Ser Ser Asn Leu Asp Gln Ala Gln Val Thr Glu Ser Arg Met 270 ~ 275 280 ACT CTT GGG ACG CAC TGC ATC AAC CCC ATC TAT~GCC TTT lass ATG TGC ATC
Thr Leu Gly Thr 8is Cys Ile Asn Pro Ile Tyr AIa Phe Met Cys Ile 285 ~ 290 295 GTC GGG GAG TTC AGA TliC CTC TTA GTC TTC CAA AAG CAC I203 AAG AAC TTC ' Val Gly Glu Phe Arg Tyr heu Leu Val Phe G,ln Lys I:ys Asn Phe 8is-300 305 . 310 315 ATT C,CC AAA TTC TGC TGC TGT TCT ATT CA6i CA1~~ GA6 I25I
CfC AAF1 ' TTC GCT
Ile AIa Zys Fhe Cys Cys Cys Ser Lle 61r~ Gln Glue Arg Lys Phe Ala CCC GAG C6A AGG TCA TAC ACC.CGA TCC GGG GAG CAG GAPS1299 GCA GTT ACT .
Pro Glu~Arg Ser Ser Tyr Thr Arg Ser GIg Glu GI~i Ala Va3. Thr Glu ATA TCT GTG GGC TTG~ TGAC~CGGAC TCAAGTGGGG TGGTGA~CC1~ GTCA6'AGTTG ~~x354 Ile Ser Val Gly Leu . ' TGCl~CATGGC TTAG~'TTTCA, TACACAGCCT GGGCTGGGGG TGGGGTGGAA GAGGTCTTTT 1414 (21' INP~5R2~tFtTION FCR SEø IET ~tQ:2':
( i a SEQUENCE CF~tPtCTERISTICS
(A) LENGTI#: 352 amino acid (B~ TYPE: amino acid (D) TOPi'~Ltt'~GY: linear ( ii ) MO1.ECDLE TYPE : protein (xi~ SEQQE1~ICE DESCRIPTION: SEQ ID N0:2:
Met Asp Tyr Gln Va3. Sex Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thx 1 S 10 ' 15 -t~T_ .
Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe~Ile Phe Gly Phe Val Gly.Asn 35 40 . 45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Gln Arg Leu GIu Ser Met Thr Asp Ile Tyr Leu Leu l~sn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 . '- 75 80 Leu Thr Val Pro Phe Trp Ala Bis Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 .95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly ?~eu Tyr Phe Ile GZy Phe 100 105 ~ 110 ' Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val 8is RIa Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Yal Val Thr Ser Val Ile Thr Txp Val Val Ala Val Phe A'La Ser ?~eu Pro Gly Ile Ile phe Thr Arg Ser Gln Lys Glu Gly Leu 8is Tyr 165 I70 . 175 Thr Cys Sex Ser Iiis Phe Pro Tyr Ser GIn Tyr Gln Phe~Trg Lys Asr~
180 185 ' 190 Phe Gln Thr Leu T.ys IIe Val Ile Leu Gly Leu Val Leu Pra~ Leu Leu X95 ~ 200 205 Yal Met Val Ile Cys Tys Ser GIy Ile Leu Lys Thr Leu Leu Arg Cys 210 21~ 22E? .
Arg Asn Glur ~ys Lys Arg 8is Arg Ala VaEI ArQ feu 31e Phe Thr Ile 225 230 235 244' Met IIe Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu . 245 250 255 .
Leu .~~s~x Thr Phe Gln Glu PFse Phe Gly Leu Asn Asn Cys Ser Ser Ser Ast~ l~rg Leu asp Gln AIa Met G~.n t~'al Thr Glu Tar. Leu Gly Met Thr 2'15 . 28~T 285 ibis Cys Cys IIe Asn Pro Ile Ile Tyr Ala Phe Val fly 6Iu Lys Phe Arg-Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile AIa I:ys Arg Phe Cys Lys Cys Cys Ser I'le Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly Glu Gin Glu Ile Ser Val G1_y Leu 3~0 - . 345 350 (2) INFORMATION FOR SEQ ID N0:3:
'(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
(2) INFORMATION
FOR SEQ
ID N0:4:
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH: 29 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4.
(2) INFORMATION FOR SEQ ID NO:S: , . (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) ' MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S: .
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 61 BASE PAIRS ..., (B) TYPE: NUCLEIC ACID _ (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CTAGCTCGAG TCAAGCGTAG TCTGGGACGT.CGTATGGGTA GCACAAGCCC ACAGATATTT 60 C . 6I
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
(2) INFORMATION FOR SEQ ID N0:8:
(i.) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29 BASE PAIRS
($) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 344 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly A1a Pro Cys His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro 20 25 30 ..
Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro Leu Trp AlawHis Ser Ala Ala Asn Glu Trp Val 80 ~ 85 90 Phe Gly Asn Ala Met Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr_Phe Gly Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp 110' 115 ~ 120 Arg Tyr Leu Ala Ile Val His Ala Val Phe Ala Leu Lys Ala Arg ' CA 02562162 2006-10-20 -5 ~-Thr Val ValThr SerVal IleThr TrpLeu Val Thr Phe Gly Va1 Ala Val Val ProGly IleIle PheThr LysCys Gln Phe Ala Ser 155 I60 ~ 165 Lys Glu SerVal Tyr ValCys GlyPro TyrPhe ProArg Gly Asp 1~0 I75 180 Trp Asn PheHis Thr IleMet ArgAsn IleLeu GlyLeu Val Asn Leu Pro LeuIle Met ValIle CysTyr SerGly IleLeu Lys Leu 200 ~ 205 - 210 Thr Leu ArgCys Arg AsnGlu LysLys ArgHis ArgAla Val Leu Arg Val PheThr Ile MetIle ValTyr PheLeu PheTrp Thr Ile Pro Tyr IleVal Ile LeuLeu AsnThr PheGln GluPhe Phe Asn Gly Leu AsnCys Glu SerThr SerGln LeuAsp GlnAla Thr Ser Gln Val GluThr Leu GlyMet ThrHis CysCys IleAsn Pro Thr rle Ile AlaPhe Val GlyGlu LysPhe ArgSer LeuPhe Eiis Tyr Ile Ala GlyCys Arg IleAla ProLeu GlnLys ProVal Cys Leu Gly Gly GlyVal Arg ProGly LysAsn ValLys ValThr Thr Pro 320 _ 325 330 Gln Gly LeuAsp Gly ArgGly LysGly LysSer IleGly Leu . 340
Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling. of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.
Chemokines, also referred to as intercriae cytokines, are a subfamily of structurally and functionally related cytokines . These molecules are 8-10 kd in size . In general, chemokines exhibit 20~c to 75~ homology at the amino acid level and are characterized by four conserved cysteine~
residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta.
In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the "C-g-C"
subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the "C-C" subfamily. Thus far, at least nine different members of this family have been identified is humans.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblaets. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histaiaine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of coatplement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflam~aation, certain chemokines have bees shown to exhibit other activities . For examcple, macrophage inflanrcc~aatory protein 1 tMIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4 ) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells .
In light of the diverse biological activities, it is not surprising that che~nokiaes have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
In accordance with one aspect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present inveatioa'are of human origin.
In accordance with aaother~ aspect of the present invention, there are. provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as aatisense- analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect of the present imreation, there are provided processes for producing such receptor polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences encoding the receptor polypeptides of the present invention, under conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.
In accordance with yet a further aspect of the present invention, there are provided antib.~dies against such receptor polypeptides.
In accordance with another aspect of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of the receptor polypeptides of the present invention.
In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in stinn~lating haematopoiesis, around healing, coagulation, angiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-imaname diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.
In accordance with another aspect of the present invention there is provided a method of administering the receptor polypeptides of the present invention via gene therapy to treat conditions related to underexpression of the polypeptides or underexpression of a ligand for the receptor polypeptide_ In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and Ig$-m;ediated allergic reactions; prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosinophilic syndrome.
In accordance with yet another aspect oft. the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.
Tn accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the~present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding such polypepti.des, ~ for .in' vitro purposes related. ' to scientific research, synthesis of DNA and manufacture of DNA vectors.-These and other aspects of the present invention should be apparent to those skilled~in the art from the teachings herein.-The following drawings acre illustrative of embodiments of the invention and are not meant to limit the scope.of the invention as encompassed by the_claims.
Figure 1 shows the cDNA sequence and the. corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention. the tandard one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequ~ncer (~..pplied Biosystems, Inc.).
Figure 2 illustrates an amino acid alignment of the G-protein chemokine receptor~of the present invention and the human MCP-I receptor.
In accordance with an aspect of the preser_t invention, there is provided an isoxated nucleic acid (polyizucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID N0:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 97183 with the Rmerican Type Culture Collection, 12301 Parklawn Drive, Rockv?11e, Mary?and, 20852, United States of America, on June ~, 1995.
The polynucleot~~.de of this invention was discovered in a cDNA library derived from human monocytes. It is stiucturallv related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 352 amino acid residues. The protein exhibits the highest degree of homology to a human MCP-1 receptor with 70.1 %
identity and 82.9 % similarity over a 347 amino acid stretch.
The polynucleotide of the present invention may be is the form of RNA or in the forca of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DN~.1 may. be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (S$Q ID
NO:1) or that of the .deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the-same mature polypeptide as the DNA of Figure 1 (S8Q ID N0:1)~
or the deposited cDl~.
.The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by~the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for- the mature palypeptide and additional-coding sequence such as a transmembraae (T1~1')- or intro-cellular domain; the coding sequence for the . mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence far the mature polypeptide. ' Thus, the term "polynucleotide encoding a polypeptide"
encooapasses a polynucleotide which includes only codir~g sequence for the polypeptide as well as-.a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the herei.nabove described polyaucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amigo acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. T~.
variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (S$Q ID N0:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as .variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SBQ~ID
NO:~) or the polypeptide encoded by the cDI~ of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the.polyaucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown is Figure 1 (S$Q ID-NO:~)_or of the coding sequence of the deposited clone. As known is the art, as allelic variant is as altezaate form of a polyaucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide_ ' The polyaucieotides'may also encode for a soluble form of the G-protein chemokine receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved froia the TM and intracellular domain of the full-leagth poiypeptide of the present invention.
The polynucleotides of the present~invention may also have the coding sequence fused~in frame to a marker sequence which allows for purification of the polypeptide of the present imrention_ The marker sequence may be a hexa-histidine tag supplied by a pQB-9 vector to provide far-purification of the mature polypeptide fused to the marker is the case of a~bacterial host, or, for example, the marker sequence may be a hemagglutinin~(HA) tag when'a-mammalian _g_ host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:76: (1984)y.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (axons).
Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA~
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases . The probe may also be used to identify a cD~~iA
clone corresponding to a full length transcript and a genomic ;
clone or clones that contain the complete gene including regulatory and pranotor regions, axons, and introns. An example of a screen coomprises isolating the coding region of the gene by using the laio~rn DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, ;.geaomic DATA or mR~ to determine which members of the library the probe hybridizes to.
The present invention further relates to polynucleotides which hybridize to the hereiaabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The-present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereiaabove-described polynucleotides. As herein used, the terra "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides _9-which hybridize to the herei.na.bave described polynucleotidE
in a preferred embodiment encode polypeptides which either retain substantially the same biological function yr activity as the mature polypeptide encoded by the cDl~s of Figure 1 (SBQ ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and awre preferaGbly at least 50 bases which hybridize to a polynucleotide'of the present imrention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SBQ ID I~IO:1, for ~ exan~le, for recovery of the polyaucleotide or as a diagnostic probe or as a PQ2 primer.
Thus, the present invention is directed to polynucleotides having at~ least a 70~ identity, preferably at Least 90~ and more preferably at least a 95~ identity to a polynucleotide which encodes the polypeptide of SBQ ID N0:2 as well as fraga~eats thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polyaucleotides.
The deposits) referred to herein will be maintained under the terms of, the Budapest Treaty on the International Recognition of the Deposit of Micro-organistas for purposes of Patent Procedure. These deposits ajre provided merely as convenience to those of skill ia-the art and are not an adatission that a deposit is required.
The segueace of the polynucleotides contained in the deposited materials, as well. as the axaino acid sequence of the polypeptides encoded thereby, are controlling in the event of nay conflict.
with~any description of~sequences herein. A license utay be required to make, or sell the deposited materials, and no such license is hereby granted..
_1Q_ The present invention further relates to a G-protein chemokine receptor polypeptide which has the deduced amino acid sequence of Figure 1 (SBQ ID N0:2) or which has the amino acid sequence encoded by the deposited'cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The terns ~fragment," "derivative" and ~anaiog~ when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e_ functions as a G-protein chemokine receptor, or retains the ability to bind the ligand or the receptor even though_the polypeptide does not function as a G-protein chemokine receptor, for example, a soluble form of the receptor. An analog includes a proprotein~which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polyQeptide of Figure 1 (SNQ ID N0:2) or that encoded by the deposited cDI~ may be (i) one in which one -or more of the am;.ao acid residues are substituted with a conserved or non-consezved amino acid residue (preferably a conserved amino acid residue)~and such substituted amino acid residue may or may not be one encoded by the genetic code, or <ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a comcpound to increase the half-life of the- polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide or (v) one in which a fragment of tie polypeptide is soluble, i.e. not membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives and analogs are deeated to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The polypeptides of the present invention include the polypeptide of SBQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably a 70% identity) to the polypeptide of SBQ ID N0:2 and more preferably a 90% similarity (more .
preferably a 90% identity) to the polypeptide of SBQ ID N0:2 and still more preferably a 95% similarity (still more preferably a 90% identity) to the polypeptide of S$Q ID N0:2 and to portions of such polypeptide with such portion of the poiypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The tezm "geese" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons)_ The term "isolated° means that the material is removed from its original environment (e. g., the natural environment if it is naturally occurring). Por example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from s~aae or all of the coexisting ~aaterials is the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a co~o~position, and still be isolated in that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SSQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at least 70% identity) to the polypeptide of S8Q ID N0:2 and more preferably at least 90%
similarity (more preferably at least 90% identity) to the polypeptide of S$Q ID N0:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SBQ ID N0:2 gad also iaciude portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amigo acids.
~ ~o~ ~ the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second po3ypeptide.
Frac~nents or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides: Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The present invention also relates to vectors which include polyaucleotides of the present invention, host cells which are genetically engineered with vectors 'of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating.
promoters , selecting transfo~mants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant tec~iques. Thus, for example, the polyaucleotide may be included in nay one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of Sv40; bacterial plasmids; phage DNA;
bacuZovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral D1~ such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicabie and viable in the host.
The appropriate DNA seguence may be inserted into~the vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction endonuclease sites) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequences) (promoter) to direct mRNA synthesis. As representative examples of such pramoters, there may be mentioned: LTR or SV4Q promoter, the E. coli. lac or t~, the phage lambda P~
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their vizuses.
The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in B. coli.
The vector containing the appropriate DID sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to traasforar an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, StreDtonHrces, SalaaonelZa tvnhimurium; fungal cells, such as yeast; insect cells such as Drosonhila and cetera animal cells such as CEO, COS or Hooves melanoma; adenovirus;
plant cells, etc. The selection of an appropriate host is deemed to.be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs co~ac~prise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably _Z~_ linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are comseercially available. The following vectors are provided by way of example. Bactei_al: pQ$70, pQB60, pQ$-9 (Qiagen), pbs, pDlO, phagescript, psiXi74; pbluescript SIC, pbsks, pl~8A, pNXl6a, pN818A, pNH46A (Stratagene); ptrc99a, pKR223-3, pKR233-3, pDR540, pRITS (Pharmacia). Bukaryotic: pWLN80, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3; pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable aad viable in the host.
Promoter regions can be selected from aay desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL aad trp.
Bukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, aad mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level. of ordinary skill in the art.
In a further embodiment, the_present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaxyotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DSAB-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (198x)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from ' the Did constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).
Transcription -of the DIZA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Bnhancers are cis-acting elements of DID,, usually about from 10 to 300 by that act on apromoter to increase its~transcription.
Examples including the SV40 eahancer on the late side of the replication origin by 100 to 270, a cytoa2egalovirus early promoter eahancer, the polyoma exl~ancer on the late side of the. replication origin, and adeziovirus enbancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae T1ZP3 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and ter~aination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular meditaa. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization~or simplified purification of expressed . recombinant product.
Useful expression vectors for bacterial use are constxucted by inserting a structural DNA seguence encoding a desired protein together With suitable translation initiation and tezmination signals in operable reading phase with a functional promoter. The vector will.comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts' for transformation include B. coli, Bacillus subtilis, Salmonella tvahimurium and various species within the ~ genera Pseudomoaas, Streptomyces, and .Staphylococcus, although others may also be employed as a~
matter of choice. ~ ' As a representative but noalimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin~of replication derived from comsterczally available plasmids cocaprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such c~ercial vectors include, for eacample, pKK223-3 (Pbar<nacia Fine Chemicals, Uppsala, Sweden) and G8M1 (Promega Biotec, Madison, WI, tTSA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e. g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, diszupted by physical or chemical means, and the resulting crude extract retained for further purification.
_18_ Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
various mammalian cell culture systems can also be employed to express recombinant protein. fixamples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C12?, 3T3, C80, Fieha and BHK cell lines. Mammalian expression vectors will coa~rise an origin of replication, a suitable promoter and eahancer, and also any necessary ribosa~ne binding sites, polyadeaylation site, splice donor and acceptor sites, transcriptional termination segueaces, and 5' flanking nontranscribed sequences. D1~1 sequences derived from the SV40 splice, and polyadeaylation sites may be used to provide the required nontranscribed genetic elements.
The G-protein che~xaokine receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial methionine amino acid residue.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.
The G-protein chemokine receptors of t3ie present im~ention may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide of the present invention .
In general, such screening procedures involve providing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, yeast, drosophila or E. Cola. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the G-protein chemokiae receptor. The expressed receptor is then contacted with a test compound to obse3cve binding, stimulation or inhibition of a functional response.
One such screening procedure involves the use of ~aelanophores which are traasfected to express the G-protein chemokine receptor of the present invention. Such a screening technique is described in PCT WO 92 / 01810 published February 6, 1992.
Thus, for example, such assay may be employed for screening for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.
The screen may be employed for determining a compound which activates the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.
Other screening techniques include the use of cells which express the G-protein chemokine receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (October 1989). For example, compounds may be contacted with a cell which expresses the receptor polypeptide of the present imrention and a second messenger response, e.g.-signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.
Another such screening technique involves introducing RtsA encoding the G-protein chemokiae receptor into 8enopus oocytes to transiently express the receptor.. The receptor oocytes may then be contacted with the receptor ligand and a cooa~pound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for caa~pounds which are thought to inhibit activation of the receptor.
Another screening technique involves expressing the G-protein chemokine receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embzyonic kidney cells, etc. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.
Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting .a eukaryotic cell with DNA encoding the G-protein chemokine receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the coarpound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
An antibody may antagonize a G-protein chemokine receptor of the present invention, or in so~e cases an oligopeptide, which bind to the G-protein chemokiae receptor but does not elicit a second messenger response such that the activity of the G-protein chemokine receptors is prevented.
Antibodies include anti-idiotypic antibodies which recognize unique determinants generally associated with the antigen-binding site of as antibody. Potential antagonist compounds also include proteins which are closely related- to the Iigaad of the G-protein chemokine receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein chemokine receptor elicit no response.
An aatisease construct prepared through the use of aatisense technology, may be used to control gene expression through triple-helix formation or aatisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DTiA or RIB. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an aatisease RNA oligoaucleotide of frost about 1f to 40 base pairs in length. A DNA oligonucleotide is designed to be coanplemeatary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988):
and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of G-protein chemokine receptor. The antisense RNA oligonucleotide hybridizes to the ~aaRNA in vivo and blocks translation of mRNA
molecules into G-protein coupled receptor (antisense - Okano, J. Neurochem.. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Baton, FI: (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA
may be expressed ~En vivo to inhibit production of G-protein chemokine receptor.
A small molecule which binds to the G-protein chemokine receptor, making it inaccessible to ligands such that nozmal biological activity is prevented, for example small peptides or peptide-like molecules, may also be used to inhibit activation of the receptor polypeptide of the present invention.
A soluble f ozln of the G-protein chemokiae receptor, a . g .
a fragment of the receptors, may be used to inhibit activation of the receptor by binding to the ligand to a polypeptide of the present invention and preventing the ligand from interacting with membrane bound G-protein chemokiae receptors.
The com~pouads which bind to and activate the G-protein chempkine receptors of the present invention may be employed to stite haematopoiesis, wound healing, coagulation, aagiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-im~awae diseases, parasitic affections, psoriasis, and to stimulate growth factor activity.
The compounds which bind to and inhibit the G-protein chemokine receptors of the present invention may be employed to treat allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and Ig$-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis shock and hyper-eosinophilic syndrome.
The compounds may be employed in combination with a suitable pharmaceutical carrier. Such'compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should ~ suit the mode of administration.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of. the pharmaceutical compositions of the invention. Associated with such containers) can be a notice in the fozm prescribed by a governmental agency regulating the manufacture, use or sale. of pharmaceuticals or biological products, which not~.ce reflects approval by the agency of _asanufacture, use or sale for human administration_ In additio'~n, the co~apouads. of the present invention may be employed in conjunction with other therapeutic coxapounds.
The pharmaceutical ,cocapositions may be ac~ainistered in a convenient manner such as by the topical, intravenous, iatraperitoaeal, iatramuseular, subcutaneous, iatraaasal or 3atradermal (applicable) routes. The pharmaceutical compositions are administered in as amount which is effective for treating and/or prophylaxis of the specific indication.
In general, the phaanaceutical compositions will be administered in an amount of at least about ZO ~eg/kg body weight and in nmost cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day.
In most cases, the dosage is from about 10 ,ug/kg to about 1 mg/kg body weight daily, taking into account the routes of adnttnistratioa, symptoms, etc., The G-protein chemokine receptor polypeptides and antagonists or agonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as ~gene therapy.~
Thus, for example, cells from a patient may be engineered with a polyaucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA
encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide is vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the preseat invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
R,etraviruses frown which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloaey Marine Leukemia Vizus, spleen necrosis vizus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis vines, gibbon age leukemia virus, human immunodeficiency virus, adenovirus;
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
In one embodiment, the retroviral plasmid vector is derived from Moloney Marine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (HIV) promoter described in Miller, et al., $~otechnioues, Vol. 7, No. 9, 980-990 (1989) , or any other promoter (e. g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a. suitable promoter. Suitable, prou~ot~rs which may be employed include, but are not limited to, adeaoviral promoters, such as the adenovirai major late proafoter; or hetorologous promoters, such as the cytomegalovirus (City) propaoter; the respiratory syncytial virus (RSV) praanoter; inducibie~promoters, such as the MMT promoter, the metallothionein proiaoter; heat. shock promoters; the alhumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thyad.dine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described).; the ~B-actin promoter; and human growth hormone promoters . The proutoter also ~ may be the native proamoter which controls the genes encoding the polypeptides.
The retroviral plas=aid vector is employed to transduce packaging cell lines to foza~ producer cell lines. Bx,amples of packaging cells which may be transfected include, but are not limited to, the PSSOl, PA337, ~-2, ~-AM, PA12, T19-14X, VT-39-I7-Fi2, s~CRB, ~C'RIP, GP+E-.86, GP+eavAml2, and DAN cell lines as described in Miller, Human Gene Theratw, vol. 1, pgs. 5-14 (1990) .
The vector may transduce the packaging cells through any means laiown in the art. Such means include, but are not limited to, electroporation; the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral 'plasmz.d vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequences) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either za vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequences) encoding the polypeptide. 8ukaryotic cells which may be transduced include, but are not limited to, embryoaic stem cells, embryonic carcinoma . cells, as well as hema_topo~:etic. stem . cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, aad bronchial epithelial cells.
The present invention also provides a method for determining whether a ligand not kaown to be capable of binding to a G-protein cheruokine receptor can bind to such receptor which comprises contacting a mammalian cell which expresses a, G-protein chemokine receptor-,with the ligand under conditions percaitting binding of ligands to . the G-protein chemokine receptor, detecting the presence of .a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein chemokine receptor.
The systems hereinabove described-for determining agonists and/or antagonists may also 'be employed for determining ligaads which bind to the receptor.
This invention also provides a method of detecting expression of a G-protein chemokine receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA coding for the receptor which comprises obtaining total mRHA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression of the receptor by the cell.
The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. . These related receptors .rnay be identif led by hortnology to a G-protei a chemokine receptor polypeptide of the present invention,-by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the chemokine receptor.polypeptides of the present invention.
Fragments of the genes may, be ~ used as a hybridization probe for a cDi~.1 library to isolate other genes which have a high sequence similarity to the genes of- the present invention, or which have similar biological activity. Probes of this type are at least 20 bases, preferably at least. 3.0 bases and angst preferably at least 50 bases or more. The probe may also be used to identify a cDI~1 clone corresponding to a full length'transcript and a genomic clone or clones that contain the caa~plete gene of the present invention including .regulatory and promoter regions, exons and introas .
An example of a screen of tb.is type comprises isolating the coding region of the gene by using the known DNA sequence to syathesize~ an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the. genes of the present invention are used to screen a library of human cDNA, genosaic DNA or mRNA to deter~ni.ae which members of the library the probe hybridizes to.
The present invention also contecuplates, the use of the genes of the present invention as a diagnostic, for example, score diseases result from inherited defective genes. .These genes can be detected by cooaparing the sequences of the defective gene with that of a normal one. Subsequently, one can verify that a "mutant" gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expression in a functional assay system (e. g., colorimetric assay, expression on MacConkey plates, c~rplementation experiments, in a receptor deficient strain of ~IC293 cells) as yet another means to verify or identify mutations. Once "mutant" genes have been identified, one can they screen population for carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techaigues. Nucleic acids used for diagnosis may be obtained froaa a patient' s cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material_ The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR
(Saiki, et al., N~.tu~, 324:163-166 1986) prior to analysis.
RNA or cDNA may also be used for the same purpose. As an example, PCR primers como~plimentazy to the nucleic acid of the instant invention can be used to identify and analyze mutations in the gene of the present invention. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to. radio labeled RNA of the invention or alternatively, radio labeled aatisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting te~peratures. Such a diagnostic would be particularly useful for prenatal or eves neonatal testing.
Sequence differences between the reference gene and "mutants" may be revealed by the direct DNA seqeiencing method. in addition, cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For exa~aa~ple, a sequence primer is used with double stranded PCR
product or a single stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radio labeled nucleotide or h:
an autoioaatic sequencing procedure With fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at specific locations may also be revealed by nucleus protection assays, such RNase and S1 protection or the chemical cleavage method (e.g. Cotton, et al. , PI~~~g. USA, 85:4397-4401 1985) .
In addition, sane diseases are a result of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be used as a reference to identify individuals expressing a decrease of functions associated with receptors of this type.
The present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the G-proem chemokiae receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well kaoarn to those of skill in the art and include radioimmvaoassays, competitive-binding assays, Western blot analysis and preferably as BLISA assay.
An BLISA assay initially coa~rises preparing an antibody specific to antigens of the G-protein chemokine receptor polypeptides, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzycae. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any G-protein chemokine receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody'is washed ou~ with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to G-protein chemokine receptor proteins. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of G-protein chec~okine receptor proteins present in a given volume of patient sample when cort~ared against a standard curve.
The seguences of the present invention are also valuable for chroancso~ne identification. The sequence is specifically targeted to and can hybridize with a particular location on as individual human chromoso~ane.. Moaceover, there is a current need for identifying particular sites on the chromosome . Few chro~some marking reagents based on actual sequence data (repeat polymo~cphisms) are presently available for marking chro~nosoma,,l location. The mapping of DNAs to chromosomes according to the present invention is an important f first step in corzelating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from' the cDNA.
Computer analysis of the cDNA is used to rapidly select primers that do not span more than one axon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell -3~-hybrids containing iadividtial human chro~awsomes. Oniy.those hybrids containing the human gene corresponding to the primer will yield an amplified fragaaent.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large geaomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in s~ttu hybridization, prescreening With labeled flow-sorted chromosomes and preselection by hybridization to construct chromaosome specific-cDl~ libraries.
Fluorescence ~fn situ hybridization (FISH) of a cDN~I
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York ( 1988 ) .
Once a sequence has been mapped to a precise chromosomal location, the physical position, of the sequence on the chroaaosome can be correlated with genetic map data. Such data are found, for example, in v. McRusick, Meadelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have bees mapped to the same chromosomal region are they identified through linkage analysis (coinheritarice of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals . If a mutation is observed in sa~ae or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes z megabase mapping resolution and one gene per 20 kb) .
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an im~nuaogen to produce antibodies thereto. These 'antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be~
obtained by direct injection of the polypeptides into an animal or by ad~ainisterinc the polvpeptides to an animal, preferably a nonhuman . The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment,of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human E-cell hybridama. technique (Rozbor et al : , 1983 , Imaaunology Today 4 : 72 ) , and the BBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies tll.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, tram~Anic mice may be used to express humanized antibodies to imvanunogenic polypeptide products of this imrention.
The present i~m~ention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate understanding of the following examples certain freguently occurring methods and/or terms will be described.
"Plasmids~ are designated by a lower case p preceded aad/or followed by capital letters and/or numbers. The starting plasmids herein are either co~mmnercially available, publicly available on an unrestricted. basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~cg of plasmid or DNA
fragment is used with about 2 units,of enzyme in about 20 ~cl of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~cg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzyr~aes are specified by the manufacturer . Incubation times of about 1 hour at 3 7~' C are ordinarily used, but may vary in accordance with the supplier s instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide geI described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be che~aically synthesized. Such synthetic oligonucieotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP is the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomaplished using known buffers and conditions with 10 units to T4 DNA lipase ("lipase") per 0.5 ycg of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Bb, A., Virology,.52:456-457 (1973).
Exa~le 1 Bacterial Bxflression and Purification of HDGNR10 The DNA sequence encoding for 5Z~R10, ATCC # _ is initially amplified using PCR oligonucleotide primers corresponding to theca 5' and sequences of the processed ~RlO protein (minus the signal peptide sequence) and the vector sequences 3' to the F~GNR10 geese. Additional r nucleotides corresponding to FmGYRIO were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence S' CGGAATTCCTCCATGGATTATCAAGTGTCA 3' contains an BcoRI restriction enzyme site followed by 18 nucleotides of H~N'RIO coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5~' CGGAAGCTTCGTCACAAGCCCACAGATAT 3' contains complementary sequences to ~ HindIII site and-is followed by z8 nucleotides of F~GNR10 coding sequence. The restriction enzyme sites correspond to the restr~.ctioa enzyme sites on the bacterial expression vector pQE-9 .(Qiagen, inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQB-9 encodes antibiotic resistance (A~up') , a bacterial origin of replication (ori) , as IPTG
regulatable promoter operator (P/0), a ribosome~binding site (RBS), a 6-His tag_and restriction enzyme sites. pQE-9 Gras then digested- with EcoRI and HindIII. The amplified t"
sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding.for the histidine tag and the RBS. The ligation mixture was then used to transforiu E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described zn Saiabrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (3989). M15/rep4 contains paultiple copies of~the plasmid pRFP4, which expresses the lacl repressor and also confers kanaa~ycin resistance (Xan°) .
Tzansfortaants are identified by their ability to grow on L8 plates and an~picillin/kanamycin resistant colonies were selected. Plasaaid , DNA was isolated and confizzaed by restriction analysis. - Clones containing the desired constnzcts were grown overnight (0/N) in liquid culture in Ia8 media supple~oaented with both Amp (Z00 ug/zal) and Kan (25 ug/ZN.). The O/N culture is used to inoculate a large culture at a ratio of 1.100 to- 1:250. The cells were grown tq an optical density 600 (0.D.°°°) of between 0.4 and 0.6.
IPTG
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing. the ?/0 leading to increased gene expression. Cells were grown an extra 3 to 4 hours .
-36=
Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCi. After clarification, solubilized HDGNR10 was purified from this solution by chromatography on a Nickel-Chelate~
column under conditions that allow for tight binding by groteins containing.the 6-His tag. Hochuli, E. et al., J.
Chromatography x11:17?-I84 (198x). HDGNR10 was eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine KC1, lO.OmM.sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione ~ (oxidized) . After incubation iri this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.
example 2 The expression .of plasrnid, HDGNR10 Iii is :derived front a vector pcDNAI/AmpM(Invitrogen) containing: 1) ~SV40 origin of replication, 2) ampicillin resistance gene, 3) E_coli.
replication origin, a) CMV promoter followed by a polylinker region, a SVaO intron and polyadenylation site. A DNA
fragment encoding the entire HDGNR10 precursor and a HA tag fused=in frame to its 3~ end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
.Niman, R. I~eighten,. A Cherenson, M. Connolly; and R. Lerner, 198x, Cell 37, 7~7) . The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction, strategy is described as follows:
The DNA sequence encoding for HDGNR10, ATCC No. 97L83, was constructed by PCFt using two primers: the S~ primer 5' GTCC
AAGCTTGCCACCATGGATTATCAAGTGTCA 3' and contains a HindIIT sit~-followed by 18 nucleotides of HDGNR10 coding sequence starting from the initiation colon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTt:TGGGACGTC~TATGGGTAGCACA.AGCCCACAGATATTTC
3' contains complementary ' sequences to an XhoI site, translation stop colon, HA tag and the last l8 nucleotides of the F~GNR10 coding sequence (not including the stop.codon).
Therefore, the PCR product contains a HindIII site F~GNR10 coding sequence followed by. HA tag fused in frame, a translation termination stop colon next to the HA tag, and an XhoI.site. The PCR amplified DNA fragment and the vector, pcDl~tAI/Amp, were digested with HindIII and Xhol restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning' Systems, 11099 North Torrey Pines Road, La Jolla, CA 92437) the transformed culture was - plated on atapicillin media plates and resistant ~ colonies were selected. Plasraid DNA was isolated from transfozmants and PYam~ned by restriction analysis for the presence of the correct fragment. For expression of the recoiabinant ~GNR~.O, COS cells were transfected 'with the expression vector by DBAE-DEXTRAN
method. (J. Sambrook, F. Pritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory press, (3989)). The expression of the HDGNR10 HA protein was detected by radiolabelling and iman:noprecipitation method.
(8. Harlow, D. Lane, Antibodies:. A Laboratory Manual, Cold Spring Harbor Laboratory Press, (i988)). Cells were labelled for 8 hours with '~S-cysteine two days past transfection.
Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 :nM NaCI, 1~ NP-40, 0.1~ SDS, i~~
NP-40, 0.5$ DOC, 50mM Tris, pH 7.5). (Wilson, I. et al " Id.
37:767 (1984)x. Both cell lysate and culture media were precipitated with a EA specific monoclonal antibody:
Proteins precipitated were analyzed on 15~c SDS-PAGE gels.
F.~s.~~.~tl a 3 n th v' g~grP~sion svstem The DNA, sequence encoding the full length HDGNR10 protein, ATCC No. 97183, 'was amplified using PCR
oligonucleotide primers corresponding to the 5' .and 3' sequences of the gene : ' The S' primer has the sequence 5' CGGGATCCCTCCATGGATTAT
CAAGTGTCA 3' and contains a BamHI restriction enzyme. site followed by 4 nucleotides resembling-anlefficient signal for the initiation of translation in eukaryotic cells (J. Mol.
Biol. 1987,-196, 9Q7-950, Kozak, M.), and just behind the first 18 nucleotides of the HDGNR10 gene (the initiation codon for translation is "ATG"). ' The 3' primer has the sequence 5' CGGGATCCCGCT
CACAAGCCCACAGATAT 3' and container the cleavage site for the restriction endonuclease BamHI and- 18nucleotides complementary- to the 3' non-translated sequence of the HDGNRIO gene. The amplified 'sequences were isolated from a I% agarose gel using a commercially available kit ( "Geneclean, " BIO 10I Inc . , La -Jolla, Ca . ) . The fragment was then digested with the endonuclease BarnFII -and purified as described above. This fragment is designated F2.
The vector .pRGl (modification. of pvL9~1 'vector, discussed below) is used for the expression of the HDGNR10 protein using the baculovirus expression system (for revie~r see: Summers, M.D, and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural- Experimental Statiori Bulletin No. 1555) .
This expression vector contains the stroizg polyhedrin promoter of the Autographs californica nuclear polyhedrosis virus (AcMNPV) -followed by the recognition sites for the restriction endonuclease BamFiI. The polyadenylation site of the simian virus (SV}40 is used for efficient polyadenylation. For an easy selection of recomb~~nant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed ' by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. aad Suaaners, M.D. , Virology, 170:31-39) . .
The plasmid was digested With the restriction enzyme BamHI and then dephosphorylated usiag calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1~ agarose gel as described above. This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA lipase. ls.coli HS101 cells were then transformed and bacteria identified that contained the plasmid (pBacBDGrTRIO ) with the HDC;NR10 gene using the eazyn~e BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.
~Cg of the plas-.nid pBac~GNRlO were co-transfected with i.0 ~cg of a caaamercially available liaearized baculovirus ("BaculoGold" baculovirus DNA°, Phazmingen, Saa Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
leg of BaculoGold'"' virus DNA and 5 ~g of the plasmid pBacBDGrTRIO were mixed in a sterile well of a microtiter plate containing 50 ~ul of serum free Grace's meditma (Z.ife Technologies Inc., Gaithersburg, MD). Afterwards 10 girl Lipofectin plus 90 ~Cl Grace's median were added, mixed and incubated for 15 miautes at room t~perature. Then the..
transfection mixture Was added drop wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 n~ tissue culture plate with 1 ml Grace' medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27°C. After S hours .the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf senun was added. The plate was put back into an incubator and cultivation continued at 27°C for four days.
After four days the supernatant was collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows as easy isolation of blue stained plaques. ~ (A
detailed description of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life_Technologies Inc., Gaithersburg, page 9-10) _ Four days after the serial dilution, the viruses were added to the cells, blue stained plaques were picked with the tip of an Eppendarf pipette. T3~e agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing~200 ~.l of Grace's medium.. The agar was removed by a brief centrifugation and the supernatant containing. the recombinant baculoviruses was used to infect Sf9 cells seeded in 35 moa dishes. Four days later the supernatants of these culture dishes were harvested and~then stored at 4°C.
Sf9 cells were grown in Grace's anedium~supplemented~with 10% heat=inactivated FBS_ The cells were infected with the recombinant baculovinxs V-~GNR10 at a multiplicity of infection tMOI) of 2. Six hours later the medium Was removed and replaced with SF900 II mediuui minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later S ~cCi of ~S-methionine and 5 ~.Ci 'sS cysteine (Amersham) were added. The cells were further incubated for i6 hours before they were harvested by centrifugation and the Zabelled proteins visualized by SDS-PAGE and autoradiography.
Exatrmle 4 - ExoressiQn via Gene Therap~r Fibroblasts are obtained from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10~ FBS, penicillin and streptomycin, is added.
This is then incubated at 37°C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayex is trypsinized and scaled into larger flasks.
pMV-7 (Rirsctmneier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloaey marine sarcoma virus, is digested with BcoRI and HiadII2 and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR prisoners which correspond to the 5' and 3' end sequences respectively: The 5' primer ccntains an $coRI sits and the 3' primer contains a HindIII site. $qual quantities of the Moloney murinE sarcoma virus linear backbone and the BcoRI and HindIII fragment are added together, in the presence of T4 DNA lipase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA3l~ or GP+aml2 packaging cells are grown in tissue culture' to confluent density in Dulbecco~s Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin arid streptomycin. The.MSV vector containing the ' gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (.the packaging cells are now referred to as producer cells).
Fresh ~aedia is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the_media from the producer cells. This media is removed and replaced with fresh med~,a. ~ If the titer of vixus is high, then virtually all fibroblasts will be infected and no~selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or hid.
The engineered fibroblasts are then infected into the ho$t, either alone or after~~having been grows to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
Numerous modifications and variations of the present invention are possible in light of the above~teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: HUMAN GENOME SCIENCES, INC. .
ROCKVILLE, MD 20850 UNITED STATES OF AMERICA
APPLICANT/INVENTOR: LI, Yi RUBEN, Steven M.
_, (ii) TITLE OF INVENTION: IiUMAN G-PROTEIN CHEMOKINE RECEPTOR HDGNRIO
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
(B) STREET: 1100 NEW YORK AVE., NW, SUITE 600 (C) CITY: WASHINGTON
(D) STATE: DC
(E) COUNTRY: USA
(F) ZIP: 20005 (v) COMPtTTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible - .
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,216,912 (B) FILING DATE: 06-JUN-1995 (C) CLASSIFICATIdN:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US95/07173 (8)- FILING DATE: 06-JUN-1995 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: STEFFE, ERIC K.
(B) REGISTRATION NUMBER: 36,688 (C) REFERENCE/DOCKET NUMBER: 1488.I15CA00 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 371-2600 (B)..TELEFAX: (202) 371-2540 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1419 base pairs (B) TYPE: nucleic acid (C). STRANDEDNESS: double ~ .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATDRE:
(A) NAME/FEEY: CDS
(B) LOCATION: 259._131!
(xi) SEQUE1~TCE DESCRIPTION: SEQ ID NO:1: _ GGAAGCTAGC AGCAAAGCTT CCCTTCACTA CGAAACTTCA TTGCTTGGCC CAAAAGAGAG z20 GCATTCATGG AGGGCAAC2A'AATACFsTTCT AGGACTTTAT AAAAGATCAG TTTTTATTTA 240 Met Asp Tyr 61~x Val Ser Ser Pro Ile Tyr Asp ATC AAT TAT TAT gCA TCG 6AG CCC ~'GC CCA AAA ATC AAT GTG AAG CAA . 339 Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Pro hys Ile Asn Val Lys~ Gln 15 20 ~ 25 IIe~Ala Ala'Arg Leu he~u Pro Pro heu Tyr Ser Leu Val Phe Ile Phe 30 35 - . 40 GGT TTT GTG GGC AAC ATG CTG GTC ATC CTC _AT~C CTG ATA AFiC TGC CAA ~ ' 435 Gly Phe Val Gly Asa Met Leu Val_ Ile ~.eu Ile Leu Ile Asa Cys Gln 45 54 ~ SS
AGG CTG GAG AGC ATG ACT GAC ATC TAC CTG CTC AAC CTG GCC.ATC TCT 9S3 Arg beu GIu Ser Met Thr Asg I1e Tyr Z.eu F.eu Asia Le3~ Ala Ile Ser 6f? 65 70 75 .
GAC CTG TTT TTC CTT CTT ACT GTC CCC TTC TGG ~GCT CAC 1'l~lT GCT GCC 53I
Asp Leu Phe Phe.heu l.eu- Thr VaI Pro Phe Trp Ala 8is Tyr Ala Ala '. 80 85 90 GCC CAG TGG GAC TTT GGA AAT ACA. 1$TG T'GT CAA CTC T~'G' ACA. GGG CTC ' 579' Ala Gln Trp Asp Phe Gly Asn Thr Met Cys GIr~ _ F~e~x Few TY~rr Gly heu Tyr Phe Ile Gly Phe Phe Ser Gly Tle Phe Phe IIe Ile Ixtz beu Thr lla ~~.s ~ 120 . . ~ .
ATe c~T AGG~TAC eT~ GCT ATC GTC C~~ GCT.GTG TTT GCS TTA AAA cce _. s7s ..
I2e Asp Arg Tyr Lew Rla Ile Val F~is ~ Ala ~i'af Phe F~a~ ~ ~eu ~.ys P~.a 12S ~ 134 . 135 AGG ACG GTC ACC TT'F GGG GTG GTG~ACA AGT GTG ATC ACT TG6 6TG GTG 723 Arg Thr Val Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp Pal Val Ala Va2 Phe Ala Sec heu Pro Gly Ile fle Phe Thr Arg Ser Gln Lys 16c~ lss ~ loo GAA~GGT CTT CAT TAC ACC TGC AGC TCT CAT TTT CCA TAC AGT CAG TAT 819 Glu Gly Leu His Tyr Thr Cys Ser Sex ~~is.Phe Pro Tyr Ser Gln Tyr z~7s . zeo 18s -q.s-CAA TTC TGG AAG AAT TTC CAG ACA TTA AF~G ATA GTC ATC TTG GGG CTG 867 Gln Phe Trp Lys Asn Phe Gln.Thr Leu Lys Ile Val Ile heu Gly Leu Val Leu Pro Leu leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys ACT"CTG CTT CGG TGT CGA AAT GAG AAG AAG AGG CAC AGG GCT GTG AGG 963 Thr Leu f~eu Arg Cys Arg Asn Glu r.ys Lys Arg His Arg~ Ala Val Arg CTT ATC TTC ACC ATC ATG ATT GTT TAT TTT CTC TTC TGG GCT CCC TAC ' 1011 Leu Ile Phe Thr Ire Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr .
24'O 245 250 Asn Ile Val Leu Leu Leu Asn Thr Phe Gln Glu Phe Phe~ Gly Leu Asn AGC AGG TTG
GAC CAA
GCT ATG
Asn Cys Ser Ser Asn Leu Asp Gln Ala Gln Val Thr Glu Ser Arg Met 270 ~ 275 280 ACT CTT GGG ACG CAC TGC ATC AAC CCC ATC TAT~GCC TTT lass ATG TGC ATC
Thr Leu Gly Thr 8is Cys Ile Asn Pro Ile Tyr AIa Phe Met Cys Ile 285 ~ 290 295 GTC GGG GAG TTC AGA TliC CTC TTA GTC TTC CAA AAG CAC I203 AAG AAC TTC ' Val Gly Glu Phe Arg Tyr heu Leu Val Phe G,ln Lys I:ys Asn Phe 8is-300 305 . 310 315 ATT C,CC AAA TTC TGC TGC TGT TCT ATT CA6i CA1~~ GA6 I25I
CfC AAF1 ' TTC GCT
Ile AIa Zys Fhe Cys Cys Cys Ser Lle 61r~ Gln Glue Arg Lys Phe Ala CCC GAG C6A AGG TCA TAC ACC.CGA TCC GGG GAG CAG GAPS1299 GCA GTT ACT .
Pro Glu~Arg Ser Ser Tyr Thr Arg Ser GIg Glu GI~i Ala Va3. Thr Glu ATA TCT GTG GGC TTG~ TGAC~CGGAC TCAAGTGGGG TGGTGA~CC1~ GTCA6'AGTTG ~~x354 Ile Ser Val Gly Leu . ' TGCl~CATGGC TTAG~'TTTCA, TACACAGCCT GGGCTGGGGG TGGGGTGGAA GAGGTCTTTT 1414 (21' INP~5R2~tFtTION FCR SEø IET ~tQ:2':
( i a SEQUENCE CF~tPtCTERISTICS
(A) LENGTI#: 352 amino acid (B~ TYPE: amino acid (D) TOPi'~Ltt'~GY: linear ( ii ) MO1.ECDLE TYPE : protein (xi~ SEQQE1~ICE DESCRIPTION: SEQ ID N0:2:
Met Asp Tyr Gln Va3. Sex Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thx 1 S 10 ' 15 -t~T_ .
Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe~Ile Phe Gly Phe Val Gly.Asn 35 40 . 45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Gln Arg Leu GIu Ser Met Thr Asp Ile Tyr Leu Leu l~sn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 . '- 75 80 Leu Thr Val Pro Phe Trp Ala Bis Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 .95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly ?~eu Tyr Phe Ile GZy Phe 100 105 ~ 110 ' Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val 8is RIa Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Yal Val Thr Ser Val Ile Thr Txp Val Val Ala Val Phe A'La Ser ?~eu Pro Gly Ile Ile phe Thr Arg Ser Gln Lys Glu Gly Leu 8is Tyr 165 I70 . 175 Thr Cys Sex Ser Iiis Phe Pro Tyr Ser GIn Tyr Gln Phe~Trg Lys Asr~
180 185 ' 190 Phe Gln Thr Leu T.ys IIe Val Ile Leu Gly Leu Val Leu Pra~ Leu Leu X95 ~ 200 205 Yal Met Val Ile Cys Tys Ser GIy Ile Leu Lys Thr Leu Leu Arg Cys 210 21~ 22E? .
Arg Asn Glur ~ys Lys Arg 8is Arg Ala VaEI ArQ feu 31e Phe Thr Ile 225 230 235 244' Met IIe Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu . 245 250 255 .
Leu .~~s~x Thr Phe Gln Glu PFse Phe Gly Leu Asn Asn Cys Ser Ser Ser Ast~ l~rg Leu asp Gln AIa Met G~.n t~'al Thr Glu Tar. Leu Gly Met Thr 2'15 . 28~T 285 ibis Cys Cys IIe Asn Pro Ile Ile Tyr Ala Phe Val fly 6Iu Lys Phe Arg-Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile AIa I:ys Arg Phe Cys Lys Cys Cys Ser I'le Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly Glu Gin Glu Ile Ser Val G1_y Leu 3~0 - . 345 350 (2) INFORMATION FOR SEQ ID N0:3:
'(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
(2) INFORMATION
FOR SEQ
ID N0:4:
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH: 29 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4.
(2) INFORMATION FOR SEQ ID NO:S: , . (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) ' MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S: .
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 61 BASE PAIRS ..., (B) TYPE: NUCLEIC ACID _ (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CTAGCTCGAG TCAAGCGTAG TCTGGGACGT.CGTATGGGTA GCACAAGCCC ACAGATATTT 60 C . 6I
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
(2) INFORMATION FOR SEQ ID N0:8:
(i.) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29 BASE PAIRS
($) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 344 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly A1a Pro Cys His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro 20 25 30 ..
Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro Leu Trp AlawHis Ser Ala Ala Asn Glu Trp Val 80 ~ 85 90 Phe Gly Asn Ala Met Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr_Phe Gly Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp 110' 115 ~ 120 Arg Tyr Leu Ala Ile Val His Ala Val Phe Ala Leu Lys Ala Arg ' CA 02562162 2006-10-20 -5 ~-Thr Val ValThr SerVal IleThr TrpLeu Val Thr Phe Gly Va1 Ala Val Val ProGly IleIle PheThr LysCys Gln Phe Ala Ser 155 I60 ~ 165 Lys Glu SerVal Tyr ValCys GlyPro TyrPhe ProArg Gly Asp 1~0 I75 180 Trp Asn PheHis Thr IleMet ArgAsn IleLeu GlyLeu Val Asn Leu Pro LeuIle Met ValIle CysTyr SerGly IleLeu Lys Leu 200 ~ 205 - 210 Thr Leu ArgCys Arg AsnGlu LysLys ArgHis ArgAla Val Leu Arg Val PheThr Ile MetIle ValTyr PheLeu PheTrp Thr Ile Pro Tyr IleVal Ile LeuLeu AsnThr PheGln GluPhe Phe Asn Gly Leu AsnCys Glu SerThr SerGln LeuAsp GlnAla Thr Ser Gln Val GluThr Leu GlyMet ThrHis CysCys IleAsn Pro Thr rle Ile AlaPhe Val GlyGlu LysPhe ArgSer LeuPhe Eiis Tyr Ile Ala GlyCys Arg IleAla ProLeu GlnLys ProVal Cys Leu Gly Gly GlyVal Arg ProGly LysAsn ValLys ValThr Thr Pro 320 _ 325 330 Gln Gly LeuAsp Gly ArgGly LysGly LysSer IleGly Leu . 340
Claims (9)
1. An antibody that specifically binds a human G-protein chemokine receptor HDGNR10 polypeptide.
2. The antibody of claim 1, wherein said HDGNR10 polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:2.
3. The antibody of claim 1, wherein said HDGNR10 polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:2.
4. The antibody of claim 1, wherein said HDGNR10 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:2.
5. The antibody of any one of claims 1 to 4, wherein said antibody antagonizes an activity of said HDGNR10 polypeptide.
6. Use of the antibody of any one of claims 1 to 4 to detect expression of a human G-protein chemokine receptor protein.
7. A process for detecting expression of a human G-protein chemokine receptor protein, said method comprising:
(a) contacting a protein sample isolated from a subject with the antibody of any one of claims 1 to 4; and (b) monitoring specific binding of said antibody, wherein detection of specific binding is indicative of expression of said human G-protein chemokine receptor protein.
(a) contacting a protein sample isolated from a subject with the antibody of any one of claims 1 to 4; and (b) monitoring specific binding of said antibody, wherein detection of specific binding is indicative of expression of said human G-protein chemokine receptor protein.
8. A pharmaceutical composition comprising the antibody of any one of claims 1 to and a pharmaceutically acceptable carrier.
9. Use of the antibody of claim 5 in the treatment of chronic inflammation, acute inflammation, or rheumatoid arthritis.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002216912A CA2216912A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002216912A Division CA2216912A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2562162A1 true CA2562162A1 (en) | 1996-12-12 |
Family
ID=4161544
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002562211A Abandoned CA2562211A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002216912A Abandoned CA2216912A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002562162A Abandoned CA2562162A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002562211A Abandoned CA2562211A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
CA002216912A Abandoned CA2216912A1 (en) | 1995-06-06 | 1995-06-06 | Human g-protein chemokine receptor hdgnr10 |
Country Status (1)
Country | Link |
---|---|
CA (3) | CA2562211A1 (en) |
-
1995
- 1995-06-06 CA CA002562211A patent/CA2562211A1/en not_active Abandoned
- 1995-06-06 CA CA002216912A patent/CA2216912A1/en not_active Abandoned
- 1995-06-06 CA CA002562162A patent/CA2562162A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
CA2216912A1 (en) | 1996-12-12 |
CA2562211A1 (en) | 1996-12-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6800729B2 (en) | Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor) | |
US6743594B1 (en) | Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5) | |
US20080241124A1 (en) | Human G-Protein Chemokine Receptor (CCR5) HDGNR10 | |
CA2289046A1 (en) | Human g-protein coupled receptors | |
CA2216990A1 (en) | Human g-protein chemokine receptor hdgnr10 | |
US20050266527A1 (en) | Human G-protein receptor HIBEF51 | |
EP0886643A1 (en) | Human g-protein chemokine receptor hsatu68 | |
EP1145721A2 (en) | Human G-protein chemokine receptor HDGNR10 (CCR5 receptor) | |
EP1149582A2 (en) | Human G-protein chemokine receptor HDGNR10 (CCR5 receptor). Uses thereof | |
US6338951B1 (en) | G-protein parathyroid hormone receptor HLTDG74 | |
US20020106741A1 (en) | G protein receptor HTNAD29 | |
US20080312178A1 (en) | Human G-Protein Receptor HGBER32 | |
CA2562162A1 (en) | Human g-protein chemokine receptor hdgnr10 | |
US20060014243A1 (en) | Human G-protein chemokine receptor HSATU68 | |
CA2224094A1 (en) | Human amine receptor | |
US20020086362A1 (en) | Human amine receptor | |
AU760468B2 (en) | G-protein receptor HTNAD29 | |
CA2221616A1 (en) | Human g-protein receptor hibef51 | |
CA2307709A1 (en) | Two human g-protein coupled receptors: ebv-induced gpcr 2(ebi-2) and edg-1-like gpcr | |
JP2007130021A (en) | Human g protein chemokine receptor hdgnr10 | |
JP2008086324A (en) | Human g protein chemokine receptor hdgnr10 | |
AU2003235001A1 (en) | G-Protein Receptor HTNAD29 | |
CA2210471A1 (en) | Human chemokine beta-11 and human chemokine alpha-1 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Discontinued | ||
FZDE | Discontinued |
Effective date: 20121130 |