# US 2013/0157284 A1 · Colorimetric and fluorescent proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0157284 A1 - Document ID: US20130157284A1 - Status: Published application - Kind: A1 - Application number: 13/636,499 - Filed: 2011-03-23 - Published: 2013-06-20 - Earliest claimed: 2010-03-23 - Assignees: Board of Trustees of Michigan State University - Inventors: Babak Borhan; James Geiger; Wenjing Wang; Chrysoula Vasileiou; Kin Sing Lee; Tetyana Berbasova - Web page: https://patentgrep.com/patent/US20130157284A1 ## CPC - C07K 14/47: from mammals - G01N 33/582: {with fluorescent label} - C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The invention relates to intracellular lipid binding proteins that bind retinoids and/or dye ligands and that are modified to transmit or emit light at a variety of different wavelengths. ## Claims ### Claim 1 An isolated nucleic acid encoding a modified polypeptide selected from a member of the intracellular lipid binding protein family, wherein the modified polypeptide transmits or emits light when bound to a retinoid or fluorescent dye molecule, and wherein the intracellular lipid binding protein has been modified so that an amino acid at any of positions 102-135 can form a Schiff base with a retinoid. ### Claim 2 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified polypeptide that has been modified by replacement of an amino acid at any of positions 102-135 with a lysine. ### Claim 3 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified polypeptide that has been modified by replacement of a glutamine at any of amino acid positions 107, 108 or 109 with a lysine. ### Claim 4 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified polypeptide that has been modified by replacement of an arginine at any of amino acid positions 110, 111 or 112 with a lysine. ### Claim 5 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified polypeptide that has been modified by replacement of an arginine at any of amino acid positions 131, 132 or 133 with a lysine or a glutamine. ### Claim 6 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a lysine at any of amino acid positions 39, 40 or 41 with a leucine, serine or asparagine. ### Claim 7 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a threonine at any of amino acid positions 50, 51, 52, 53, 54 or 55 with an aspartic acid, asparagine, cysteine or a valine. ### Claim 8 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a tyrosine at any of amino acid positions 59, 60 or 61 with a tryptophan, histidine, threonine, asparagine or phenylalanine. ### Claim 9 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of an arginine at any of amino acid positions 57, 58, 59 or 60 with a phenylalanine, tyrosine, tryptophan, leucine, glutamine, glutamic acid, aspartic acid or alanine. ### Claim 10 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a tyrosine at any of amino acid positions 133, 134 or 135 with a phenylalanine. ### Claim 11 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a threonine at any of amino acid positions 28, 29 or 30 with a leucine, tryptophan, glutamic acid or aspartic acid. ### Claim 12 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of an alanine at any of amino acid positions 30, 31, 32 or 33 with a tryptophan, phenylalanine, tyrosine, serine, histidine, glutamic acid or leucine. ### Claim 13 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a tyrosine at any of amino acid positions 18, 19 or 20 with a tryptophan or phenyalanine. ### Claim 14 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a glutamine at any of amino acid positions 3, 4 or 5 with an arginine, asparagine, phenylalanine, leucine, alanine, tryptophan, threonine, glutamic acid, histidine, or lysine. ### Claim 15 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a methionine at any of amino acid positions 92, 93 or 94 with a leucine. ### Claim 16 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a glutamic acid at any of amino acid positions 72, 73 or 74 with an alanine or leucine. ### Claim 17 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a glutamine at any of amino acid positions 36, 37 or 38 with a leucine, methionine or tryptophan. ### Claim 18 (depends on 1) The isolated nucleic acid of claim 1, which encodes a modified intracellular lipid binding protein that is further modified by replacement of a glutamine at any of amino acid positions 128, 129 or 130 with a leucine, lysine, glutamic acid or tryptophan. ### Claim 19 (depends on 1) The isolated nucleic acid of claim 1, wherein the modified intracellular lipid binding protein is a modified cellular retinoic acid binding protein II (CRABPII) or a modified cellular retinol binding protein II (CRBPII). ### Claim 20 (depends on 1) The isolated nucleic acid of claim 1, encoding a modified CRABPII polypeptide with amino acid sequence SEQ ID NO:46: 1 MPNXSGNWKX IRXENXEELX KVLGXNVMLR KIXVAXXXXX 41 AVEIKXEGDT FYIKXSXXXX TXEINFKVGE EFEXXTXDXR 81 PXKSLVKWES ENKXVXEQKL LKGEGPKTSW TKELTNDGEL 121 IXTXTADDVV XTXVXVRE wherein each X is independently a genetically encoded L-amino acid, a naturally-occurring non-genetically encoded L-amino acid, a synthetic L-amino acid or a synthetic D-amino acid. ### Claim 21 (depends on 1) The isolated nucleic acid of claim 1, encoding a modified CRBPII polypeptide with amino acid SEQ ID NO:47: 1 TXDXNGTWEM ESNENXEGXX KALDXDFAXR KIXVRLTXTX 41 VXDQDGDNFK XKXTXTXXNX DXDXTVGVEF DXYTKXXDNR 81 HVKALVTWEG DVLVXVXKGE KENXGXKXWI EGDKLYXEXT 121 CGDQVCRXVX KKK wherein each X is independently a genetically encoded L-amino acid, a naturally-occurring non-genetically encoded L-amino acid, a synthetic L-amino acid or a synthetic D-amino acid. ### Claim 22 (depends on 1) A hybrid nucleic acid comprising the isolated nucleic acid of claim 1 joined to a fusion partner nucleic acid that encodes a fusion partner polypeptide. ### Claim 23 (depends on 22) The hybrid nucleic acid of claim 22, wherein the isolated nucleic acid is joined in frame to the fusion partner nucleic acid. ### Claim 24 (depends on 1) An expression cassette comprising the isolated nucleic acid of claim 1 and at least one nucleic acid segment encoding a regulatory element. ### Claim 25 (depends on 1) A vector comprising the isolated nucleic acid of claim 1. ### Claim 26 (depends on 25) A vector comprising the expression cassette of claim 25. ### Claim 27 (depends on 1) A host cell comprising the isolated nucleic acid of claim 1. ### Claim 28 (depends on 27) The host cell of claim 27, wherein the isolated nucleic acid is within an expression cassette, a vector or a combination thereof. ### Claim 29 A modified polypeptide selected from a member of the intracellular lipid binding protein family, wherein the modified polypeptide transmits or emits light when bound to a retinoid or fluorescent dye molecule, and wherein the intracellular lipid binding protein has been modified so that an amino acid at any of positions 102-135 can form a Schiff base with a retinoid. ### Claim 30 (depends on 29) The modified polypeptide of claim 29, which has been modified by replacement of the amino acid at any of positions 102-135 with a lysine. ### Claim 31 (depends on 29) The modified polypeptide of claim 29, which at has been modified by replacement of a glutamine at any of amino acid positions 107, 108 or 109 with a lysine. ### Claim 32 (depends on 29) The modified polypeptide of claim 29, which has been modified by replacement of an arginine at any of amino acid positions 110, 111 or 112 with a lysine. ### Claim 33 (depends on 29) The modified polypeptide of claim 29, which has been modified by replacement of an arginine at any of amino acid positions 131, 132 or 133 with a lysine or a glutamine. ### Claim 34 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a lysine at any of amino acid positions 39, 40 or 41 with a leucine, serine or asparagine. ### Claim 35 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a threonine at any of amino acid positions 50, 51, 52, 53, 54 or 55 with an aspartic acid, asparagine, cysteine or a valine. ### Claim 36 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a tyrosine at any of amino acid positions 59, 60 or 61 with a tryptophan, histidine, threonine, asparagine or phenylalanine ### Claim 37 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of an arginine at any of amino acid positions 57, 58, 59 or 60 with a phenylalanine, tyrosine, tryptophan, leucine, glutamine, glutamic acid, aspartic acid or alanine. ### Claim 38 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a tyrosine at any of amino acid positions 133, 134 or 135 with a phenylalanine. ### Claim 39 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a threonine at any of amino acid positions 28, 29 or 30 with a leucine, tryptophan, glutamic acid or aspartic acid. ### Claim 40 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of an alanine at any of amino acid positions 30, 31, 32 or 33 with a tryptophan, phenylalanine, tyrosine, serine, histidine, glutamic acid or leucine. ### Claim 41 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a tyrosine at any of amino acid positions 18, 19 or 20 with a tryptophan or phenylalanine. ### Claim 42 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a glutamine at any of amino acid positions 3, 4 or 5 with an arginine, asparagine, phenylalanine, leucine, alanine, tryptophan, threonine, glutamic acid, histidine, or lysine. ### Claim 43 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a methionine at any of amino acid positions 92, 93 or 94 with a leucine. ### Claim 44 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a glutamic acid at any of amino acid positions 72, 73 or 74 with an alanine or leucine. ### Claim 45 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a glutamine at any of amino acid positions 36, 37 or 38 with a leucine, methionine or tryptophan. ### Claim 46 (depends on 29) The modified polypeptide of claim 29, which is further modified by replacement of a glutamine at any of amino acid positions 128, 129 or 130 with a leucine, lysine, glutamic acid or tryptophan. ### Claim 47 (depends on 29) The modified polypeptide of claim 29, which is a modified cellular retinoic acid binding protein II (CRABPII) or a modified cellular retinol binding protein II (CRBPII). ### Claim 48 (depends on 29) The modified polypeptide of claim 29, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6-28, 39-47, or a combination thereof. ### Claim 49 (depends on 29) The modified polypeptide of claim 29, which comprises a modified CRABPII amino acid sequence SEQ ID NO:46: 1 MPNXSGNWKX IRXENXEELX KVLGXNVMLR KIXVAXXXXX 41 AVEIKXEGDT FYIKXSXXXX TXEINFKVGE EFEXXTXDXR 81 PXKSLVKWES ENKXVXEQKL LKGEGPKTSW TKELTNDGEL 121 IXTXTADDVV XTXVXVRE wherein each X is independently a genetically encoded L-amino acid, a naturally-occurring non-genetically encoded L-amino acid, a synthetic L-amino acid or a synthetic D-amino acid. ### Claim 50 (depends on 29) The modified polypeptide of claim 29, which comprises a modified CRBPII amino acid sequence SEQ ID NO:47: 1 TXDXNGTWEM ESNENXEGXX KALDXDFAXR KIXVRLTXTX 41 VXDQDGDNFK XKXTXTXXNX DXDXTVGVEF DXYTKXXDNR 81 HVKALVTWEG DVLVXVXKGE KENXGXKXWI EGDKLYXEXT 121 CGDQVCRXVX KKK wherein each X is independently a genetically encoded L-amino acid, a naturally-occurring non-genetically encoded L-amino acid, a synthetic L-amino acid or a synthetic D-amino acid. ### Claim 51 (depends on 29) The modified polypeptide of claim 29, which is mixed with or complexed with a retinoid or dye ligand. ### Claim 52 (depends on 29) A fusion protein comprising the modified polypeptide of claim 29 fused to another protein. ### Claim 53 (depends on 1) A kit comprising at least one container comprising the isolated nucleic acid of claim 1, and a second container comprising a retinoid or dye ligand that binds a modified polypeptide encoded by the isolated nucleic acid, wherein the isolated nucleic acid can be within an expression cassette or vector. ### Claim 54 (depends on 29) A kit comprising at least one container comprising the modified polypeptide of claim 29 and a second container comprising a retinoid or dye ligand that binds a modified polypeptide. ### Claim 55 (depends on 1) A method of observing a target protein in vivo comprising contacting a living cell with a retinoid or dye ligand that binds a modified polypeptide encoded by the isolated nucleic acid of claim 1, wherein the cell expresses a fusion protein comprising the modified polypeptide fused in frame with the target protein. ### Claim 56 (depends on 55) The method of claim 55, wherein the dye ligand is a compound of formula I: Ring-Y—CHO wherein: Ring is an optionally substituted C5-C14 mono-, di- or tricyclic cycloalkyl, aryl or heterocyclic ring, wherein the heterocyclic ring has at least one nitrogen or oxygen ring atom, and wherein the Ring has 1-3 optional substituents that are selected from the group consisting of alkyl, halogen, alkoxy, amino and sulfhydryl; and Y is a divalent C2-C12 alkenylene chain that optionally substituted with 1-3 alkyl groups. ### Claim 57 (depends on 55) The method of claim 55, wherein the retinoid is retinal.