Chimeric GFP-aequorin as bioluminescent Ca+at the single cell level
AssigneesInstitut Pasteur, Centre National De La Recherche Scientifique
InventorsValerie Baubet, Herve Le Mouellic, Philippe Brulet
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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).
Claims52
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.
A purified polypeptide having the amino acid sequence of SEQ ID NO:
- 1.
A purified polypeptide having the amino acid sequence of SEQ ID NO:
- 2.
A purified polypeptide having the amino acid sequence of SEQ ID NO:
- 3.
A purified polypeptide having the amino acid sequence of SEQ ID NO:
- 4.
A purified polypeptide having the amino acid sequence of SEQ ID NO:
- 5.
A purified polypeptide having the amino acid sequence of SEQ ID NO:
- 6.
A purified polynucleotide having the sequence of SEQ ID NO:
- 7.
A purified polynucleotide having the sequence of SEQ ID NO:
- 8.
A purified polynucleotide having the sequence of SEQ ID NO:
- 9.
A purified polynucleotide having the sequence of SEQ ID NO:
- 10.
A purified polynucleotide having the sequence of SEQ ID NO:
- 11.
A purified polynucleotide having the sequence of SEQ ID NO:
- 12.
A polynucleotide linker having the polynucleotide sequence of SEQ ID No:
- 13.
A polynucleotide linker having the polynucleotide sequence of SEQ ID No:
- 14.
A polynucleotide linker having the polynucleotide sequence of SEQ ID No:15.
A polynucleotide linker having the polynucleotide sequence of SEQ ID No:
- 16.
A polynucleotide linker having the polynucleotide sequence of SEQ ID No:
- 17.
A peptidic linker of at least 5 amino acids and comprising the amino acid sequence of SEQ ID No:
- 18.
A peptidic linker of at least 5 amino acids and comprising the amino acid sequence of SEQ ID No:
- 19.
A peptidic linker of at least 5 amino acids and comprising the amino acid sequence of SEQ ID No:
- 20.
A peptidic linker of at least 5 amino acids and comprising the amino acid sequence of SEQ ID No:
- 21.
A peptidic linker of at least 5 amino acids and comprising the amino acid sequence of SEQ ID No:
- 22.
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.
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])n,
- 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.
A culture as deposited at the C.N.C.M. and containing the plasmid No. I-2507.
A culture as deposited at the C.N.C.M. and containing the plasmid No. I-2508.
A culture as deposited at the C.N.C.M. and containing the plasmid No. I-2509.
A culture as deposited at the C.N.C.M. and containing the plasmid No. I-2510.
A culture as deposited at the C.N.C.M. and containing the plasmid No. I-2511.
A culture as deposited at the C.N.C.M. and containing the plasmid No. I-2512.
A culture as deposited at the C.N.C.M. and containing the plasmid No. I-2513.
Dependencies are inferred from the claim text.