Chimeric GFP-aequorin as bioluminescent Ca++ reporters 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).
Claims23
1-52. (canceled)
An isolated or purified polynucleotide encoding a peptide linker of at least 5 amino acids comprising the amino acid sequence of SEQ ID NO:
- 18, SEQ ID NO:
- 19, SEQ ID NO:
- 20, SEQ ID NO:
- 21, or SEQ ID NO:
- 22.
An isolated or purified polynucleotide encoding a fusion protein of the formula:
- GFP−LINKER−AEQ:
- wherein GFP is green fluorescent protein;
- AEQ is aequorin;
- and LINKER is a polypeptide of 14-50 amino acids.
An isolated or purified polynucleotide encoding 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.
An isolated or purified polynucleotide encoding a fusion protein of the formula:
- GFP−LINKER−AEQ;
- wherein GFP is green fluorescent protein;
- AEQ is aequorin;
- and LINKER is a polypeptide of 5-63 amino acids;
- wherein the linker includes the amino acid sequence Ser Gly Leu Arg Ser [SEQ ID NO:
- 26].
An isolated or purified polynucleotide encoding a fusion protein of the formula:
- GFP−LINKER−AEQ;
- wherein GFP is green fluorescent protein;
- AEQ is aequorin;
- and LINKER is a polypeptide of 9-63 amino acids;
- wherein the 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.
An isolated or purified polynucleotide encoding a fusion protein comprising any one of SEQ ID NOS:
- 1-6.
An isolated or purified polynucleotide encoding a fusion protein comprising two bioluminescent proteins and a peptide linker, wherein said peptide linker comprises the amino acid sequence of SEQ ID NO:
- 18.
A cell comprising a plasmid selected from:
- I-2507, I-2508, I-2509, I-2510, I-2511, I-2512, and I-2513.
Dependencies are inferred from the claim text.