Claims
- 1. A polynucleotide comprising a nucleotide sequence encoding a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2) and whose emission intensity changes as pH varies between 5 and 10.
- 2. The polynucleotide of claim 1, wherein the amino acid sequence of the protein includes the substitutions S65G/S72A/T203Y/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 3. The polynucleotide of claim 1, wherein the amino acid sequence of the protein includes the substitutions S65G/V68L/Q69K/S72A/T203Y/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 4. The polynucleotide of claim 1, wherein the amino aid sequence of the protein includes the substitutions K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 5. The polynucleotide of claim 1, wherein the amino acid sequence of the protein includes the substitution H148G in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 6. The polynucleotide of claim 1, wherein the amino acid sequence of the protein includes the substitution H148Q in the amino acid of Aequorea green fluorescent protein (SEQ ID NO:2).
- 7. An expression vector comprising expression control sequences operatively linked to a polynucleotide molecule comprising a nucleotide sequence encoding a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2) and whose emission intensity changes as pH varies between 5 and 10.
- 8. A recombinant host cell comprising the expression vector of claim 7.
- 9. The recombinant host cell of claim 8, wherein the recombinant host cell is a prokaryotic cell.
- 10. The recombinant host cell of claim 8, wherein the recombinant host cell is a eukaryotic cell.
- 11. A method of determining the pH of a region of a cell comprising:
introducing into the cell a polynucleotide encoding a polypeptide including an indicator having a first fluorescent protein moiety whose emission intensity changes as pH varies between 5 and 10; culturing the cell under conditions that permit expression of the polynucleotide; exciting the indicator; and determining the intensity of the light emitted by the first protein moiety at a first wavelength, wherein the emission intensity of the first fluorescent protein moiety indicates the pH of the region of the cell in which the indicator is present.
- 12. The method of claim 11, wherein the polypeptide encoded by the polynucleotide further includes a targeting sequence linked by a peptide bond to the indicator.
- 13. The method of claim 11, wherein the amino acid sequence of the first fluorescent protein moiety includes the substitutions S65G/S72A/T203Y/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 14. The method of claim 11, wherein the amino acid sequence of the first fluorescent protein moiety includes the substitutions S65G/V68L/Q69K/S72A/T203Y/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 15. The method of claim 11, wherein the amino acid sequence of the first fluorescent protein moiety includes the substitutions K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 16. The method of claim 11, wherein the amino acid sequence of the first fluorescent protein moiety includes the substitution H148G in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 17. The method of claim 11, wherein the amino acid sequence of the first fluorescent protein moiety includes the substitution H148Q in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 18. The method of claim 12, wherein the amino acid of the targeting sequence comprises the amino terminal 81 amino acids of human type II membrane-anchored protein galactosyltransferase.
- 19. The method of claim 12, wherein the targeting sequence comprises the amino terminal 12 amino acids of the presequence of subunit IV of cytochrome c oxidase.
- 20. A kit useful for the detection of the pH in a sample, the kit comprising carrier means containing one or more containers comprising a first container containing a polynucleotide comprising a nucleotide sequence encoding a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2) and whose emission intensity changes as pH varies between 5 and 10.
- 21. The kit of claim 20, wherein the amino acid sequence of the protein includes the substitutions S65G/S72A/T203Y/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 22. The kit of claim 20, wherein the amino acid sequence of the protein includes the substitutions S65G/V68L/Q69K/S72A/T203Y/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 23. The kit of claim 20, wherein the amino acid sequence of the protein includes the substitutions K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 24. The kit of claim 20, wherein the amino acid sequence of the protein includes the substitution H148G in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
- 25. The kit of claim 20, wherein the amino acid sequence of the protein includes the substitution H148Q in the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).
Parent Case Info
[0001] This application is a continuation-in-part of currently pending U.S. Ser. No. 09/094,359 (herein incorporated by reference), which was filed Jun. 9, 1998.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under Grant No. NS27177, awarded by the National Institutes of Health. The Government has certain rights in this invention.
Continuations (1)
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Number |
Date |
Country |
Parent |
09602641 |
Jun 2000 |
US |
Child |
10457982 |
Jun 2003 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
09094359 |
Jun 1998 |
US |
Child |
09602641 |
Jun 2000 |
US |