Fluorescent protein sensors for measuring the pH of a biological sample
AssigneeUniversity of California
InventorsRoger Y. Tsien, Juan Llopis, Rebekka M. Wachter, S. James Remington
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Abstract
Disclosed are fluorescent protein sensors for measuring the pH of a sample, nucleic acids encoding them, and methods of use. The preferred fluorescent protein sensors are variants of the green fluorescent protein (GFP) from Aequorea victoria. Also disclosed are compositions and methods for measuring the pH of a specific region of a cell, such as the mitochondrial matrix or the Golgi lumen.
Claims8
A polynucleotide comprising a nucleotide sequence encoding a functional engineered fluorescent protein whose emission intensity changes as pH varies between 5 and 10, wherein said functional engineered fluorescent protein is selected from the group consisting of
- a) a protein comprising the substitutions S65G/S72A/T203Y/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- b) a protein comprising the substitutions S65G/V68L/Q69K/S72A/T203Y/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- c) a protein comprising the substitutions K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- d) a protein comprising the substitution H148G within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:
- 2), and
- e) a protein comprising the substitutions S65G/V68L/S72A/Q80R/H148Q/T203Y or S65G/S72A/H148Q/T203Y/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- where the amino acid sequence of said functional engineered fluorescent protein is at least 95% homologous to the amino acid sequence of SEQ ID NO:2.
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 emission intensity changes as pH varies between 5 and 10, wherein said functional engineered fluorescent protein is selected from the group consisting of
- a) a protein comprising the substitutions S65G/S72A/T203Y/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- b) a protein comprising the substitutions S65G/V68L/Q69K/S72A/T203Y/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- c) a protein comprising the substitutions K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- d) a protein comprising the substitution H148G within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2), and
- e) a protein comprising the substitutions S65G/V68L/S72A/Q80R/H148Q/T203Y or S65G/S72A/H148Q/T203Y/H231L within the amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2),
- where the amino acid sequence of said functional engineered fluorescent protein is at least 95% homologous to the amino acid sequence of SEQ ID NO:2.
Dependencies are inferred from the claim text.