Fluorescent protein sensors for measuring the pH of a biological sample

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 Aequora 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.

What is claimed is:  
     
         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).