Fluorescent protein sensors for measuring the pH of a biological sample
AssigneesThe Regents of the University of California, The State of Oregon acting by and through the State of Board of Higher Education on behalf of the Unviersity of Oregon
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.
Claims38
A functional engineered fluorescent protein whose amino acid sequence comprises an amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:
- 2) containing an amino acid substitution of histidine at position 148 of SEQ ID NO:
- 2 for glycine or glutamine and amino acid substitutions selected from the group consisting of:
- F64L/S65T/H231L of SEQ ID NO:2;
- S65G/S72A/T203Y/H231 L of SEQ ID NO:
- 2;
- S65G/V68L/Q69K/S72A/T203Y/H231 L of SEQ ID NO:
- 2;
- K26R/F64L/S65T/Y66W/N1461/M153T/V163A/N164H/H231L of SEQ ID NO:
- 2;
- and
- S65G/V68L/S72A/Q80R/T203Y of SEQ ID NO:2,
- and whose emission intensity changes as pH varies between 5 and 10.
A method for determining the pH of a sample comprising:
- contacting the sample with an indicator comprising a first fluorescent protein moiety whose emission intensity changes as pH varies between pH 5 and 10, wherein said first fluorescent protein moiety comprises an amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:
- 2) containing an amino acid substitution of histidine at position 148 of SEQ ID NO:
- 2 for glycine or glutamine and amino acid substitutions selected from the group consisting of:
- F64L/S65T/H231L of SEQ ID NO:2;
- S65G/S72A/T203Y/H231L of SEQ ID NO:
- 2;
- S65G/V68L/Q69K/S72A/T203Y/H231L of SEQ ID NO:
- 2;
- K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L of SEQ ID NO:2;
- and
- S65G/V68L/S72A/Q80R/T203Y of SEQ ID NO:2;
- exciting the indicator;
- and
- determining the intensity of light emitted by the first fluorescent protein moiety at a first wavelength, wherein the emission intensity of the first fluorescent protein moiety indicates the pH of the sample.
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 functional engineered fluorescent protein whose amino acid sequence comprises an amino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:
- 2) containing an amino acid substitution of histidine at position 148 of SEQ ID NO:
- 2 for glycine or glutamine and amino acid substitutions selected from the group consisting of:
- F64L/S65T/H231L of SEQ ID NO:2;
- S65G/S72A/T203Y/H231L of SEQ ID NO:
- 2;
- S65G/V68L/Q69K/S72A/T203Y/H231L of SEQ ID NO:
- 2;
- K26R/F64L/S65T/Y66W/N1461/M153T/V163A/N164H/H231L of SEQ ID NO:2;
- and
- S65G/V68L/S72A/Q80R/T203Y of SEQ ID NO:2;
- and whose emission intensity changes as pH varies between 5 and 10.
Dependencies are inferred from the claim text.