Chimeric gfp-aequorin as bioluminescent ca++ reporters at the single cell level

Abstract

A modified bioluminescent system comprising a fluorescent molecule covalently linked with a photoprotein, wherein said link between the two proteins has the function to stabilize the modified bioluminescent system and allowing the transfer of the energy by Chemiluminescence Resonance Energy Transfer (CRET).

1 . A composition comprising a purified polypeptide, wherein said composition has the functional characteristics to bind calcium ions and to permit a measureable energy, said energy depending of the quantity of calcium bound and of the quantity of polypeptides in said composition in absence of any light excitation. 
     
     
         2 . A purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6. 
     
     
         3 - 7 . (canceled) 
     
     
         8 . A purified polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 7 to SEQ ID NO: 12. 
     
     
         9 - 13 . (canceled) 
     
     
         14 . A polynucleotide linker having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17. 
     
     
         15 - 19 . (canceled) 
     
     
         20 . A peptidic linker of at least 5 amino acids and comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22. 
     
     
         21 - 27 . (canceled) 
     
     
         28 . A modified bioluminescent system comprising two bioluminescent proteins and a peptide linker according to  claim 20 . 
     
     
         29 . A modified bioluminescent system according to  claim 28 , wherein said two bioluminescent proteins comprise at least an aequorin protein. 
     
     
         30 . A modified bioluminescent system according to  claim 29  comprising the following constituents: aequorin protein and a GFP protein. 
     
     
         31 . (canceled) 
     
     
         32 . A fusion protein of the formula:
   GFP−LINKER−AEQ;
   wherein GFP is green fluorescent protein; AEQ is aequorin; and LINKER is a polypeptide of 4-63 amino acids.   
     
     
         33 . The fusion protein as claimed in  claim 32 , wherein the linker comprises 14-50 amino acids. 
     
     
         34 . The fusion protein as claimed in  claims 32  and  33 , wherein the linker comprises the following amino acids: (Gly Gly Ser Gly Ser Gly Gly Gln Ser [SEQ ID NO: 25] wherein n is 1-5. 
     
     
         35 . The fusion protein as claimed in  claim 34 , wherein n is 1. 
     
     
         36 . The fusion protein as claimed in  claim 34 , wherein n is 5. 
     
     
         37 . A fusion protein for energy transfer from aequorin to green fluorescent protein by Chemiluminescence Resonance Energy Transfer (CRET) following activation of the aequorin in the presence of Ca++, wherein the fusion protein has the formula:
   GFP−LINKER−AEQ;
   wherein GFP is green fluorescent protein; AEQ is aequorin; and LINKER comprises the following amino acids: Gly Gly Ser Gly Ser Gly Gly Gln Ser [SEQ ID NO: 25], wherein n is 1-5; and wherein the fusion protein has an affinity for Ca++ ions and a half-life of at least 24 hours.   
     
     
         38 . A fusion protein as claimed in  claim 37 , wherein the linker includes the amino acid sequence Ser Gly Leu Arg Ser [SEQ ID NO: 26]. 
     
     
         39 . A fusion protein as claimed in  claim 38 , which further comprises a peptide signal sequence for targeting the fusion protein to a cell or to a subcellular compartment. 
     
     
         40 - 41 . (canceled) 
     
     
         42 . A culture as deposited at the C.N.C.M. and containing a plasmid selected from the group consisting of Nos. 2507, 2508, 2509, 2510, 2511 2512, and 2513. 
     
     
         43 - 52 . (canceled)