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US 6,319,669 B1Granted
Modified green fluorescent proteins
AssigneeThe Regents of the University of California
InventorsRoger Y. Tsien, Roger Heim
Life of this patent
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Abstract
Modifications in the sequence of Aequorea wild-type GFP provide products having markedly different excitation and emission spectra from corresponding products from wild-type GFP. In one class of modifications, the product derived from the modified GFP exhibits an alteration in the ratio of two main excitation peaks observed with the product derived from wild-type GFP. In another class, the product derived from the modified GFP fluoresces at a shorter wavelength than the corresponding product from wild-type GFP. In yet another class of modifications, the product derived from the modified GFP exhibits only a single excitation peak and enhanced emission relative to the product derived from wild-type GFP.
Claims71
A nucleic acid molecule, comprising;
- a nucleic acid sequence encoding a functional mutant fluorescent protein whose amino acid sequence differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least an amino acid substitution at residue 65 selected from the group consisting of S65A, S65L, S65C, S65V, S65I and S65T,
- wherein said functional mutant fluorescent protein has an excitation spectrum different from an excitation spectrum of said Aequorea wild type green fluorescent protein.
A nucleic acid molecule, comprising:
- a nucleic acid sequence encoding a functional mutant fluorescent protein whose amino acid sequence differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) at least by an amino acid substitution at residue 66 selected from the group consisting of Y66H, Y66W and Y66F,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein.
A nucleic acid molecule, comprising:
- a nucleic acid sequence encoding a functional mutant fluorescent protein whose amino acid sequence differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) at least by amino acid substitution at either I167V or I167T,
- wherein said nucleic acid sequence is operatively linked to a promoter region of interest, and
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein.
A nucleic acid molecule, comprising:
- a nucleic acid sequence encoding a functional mutant fluorescent protein whose amino acid sequence differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least an amino acid substitution at S202F and T203I,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein.
A nucleic acid molecule, comprising:
- a nucleic acid sequence encoding a functional mutant fluorescent protein whose amino acid sequence differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) comprising the mutations Y66H and Y145F;
- or Y66W, M153T, V163A and N212K;
- or Y66W, I123V, Y145H, H148R, M153T, V163A and N212K;
- or Y66W, N146I, M153T, V163A and N212K,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein.
2 more elements
A method for monitoring expression of a polynucleotide sequence encoding a polypeptide of interest, comprising:
- a) providing at least one cell, comprising:
- a nucleic acid sequence encoding a fusion protein, said fusion protein comprising a polypeptide of interest and a functional mutant fluorescent protein, said functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from al amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least an amino acid substitution at residue 65 selected from the group consisting of S65A, S65L, S65C, S65V, S651 and S65T,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein,
- wherein said nucleic acid sequence encoding said fusion protein comprises a polynucleotide sequence encoding said polypeptide of interest operably linked to a polynucleotide sequence encoding said functional mutant fluorescent protein, and
- b) observing a fluorescence characteristic of ad at least one cell or said fusion protein or said functional mutant fluorescent protein.
3 more elements
A method for monitoring expression of a polynucleotide sequence encoding a polypeptide of interest, comprising:
- a) providing at least one cell, comprising:
- a nucleic acid sequence encoding a fusion protein, said fusion protein comprising a polypeptide of interest and a functional mutant fluorescent protein, said functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least an amino acid substitution at residue 66 selected from the group consisting of Y66H, Y66W and Y66F,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein,
- wherein said nucleic acid sequence encoding said fusion protein comprises a polynucleotide sequence encoding said polypeptide of interest operably linked to a polynucleotide sequence encoding said functional mutant fluorescent protein, and
- b) observing a fluorescence characteristic of said at least one cell or said fusion protein or said functional mutant fluorescent protein.
3 more elements
A method for monitoring expression of a polynucleotide sequence encoding a polypeptide of interest, comprising:
- a) providing at least one cell, comprising:
- a nucleic acid sequence encoding a fusion protein, said fusion protein comprising a polypeptide of interest and a functional mutant fluorescent protein, said functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least an amino acid substitution at either I167V or I167T,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein,
- wherein said nucleic acid sequence encoding said fusion protein comprises a polynucleotide sequence encoding said polypeptide of interest operably linked to a polynucleotide sequence encoding said functional mutant fluorescent protein, and
- b) observing a fluorescence characteristic of at least cell or said fusion protein or said functional mutant fluorescent protein.
3 more elements
A method for monitoring expression of a polynucleotide sequence encoding a polypeptide of interest, comprising:
- a) providing at least one cell, comprising:
- a nucleotide acid sequence encoding a fusion protein, said fusion protein comprising a polypeptide of interest and a functional mutant fluorescent protein, said functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least amino acid substitutions at S202F and T203I,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein,
- wherein said nucleic acid sequence encoding said fusion protein comprises a polynucleotide sequence encoding said polypeptide of interest operably linked to a polynucleotide sequence encoding a functional mutant fluorescent protein, and
- b) observing a fluorescence characteristic of at least one cell or said fusion protein or said functional mutant fluorescent protein.
3 more elements
A method for monitoring expression of a polynucleotide sequence encoding a polypeptide of interest, comprising:
- a) providing at least one cell, comprising:
- a nucleic acid sequence encoding a fusion protein, protein comprising a polypeptide of interest and a functional mutant fluorescent protein, said functional mutant fluorescent protein comprising:
- an amino acid sequence flat differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least one amino acid substitution at position 66 selected from the group consisting of Y66H, Y66W and Y66F or at position 65 selected from the group consisting of S65A, S65L, S65C, S65V, S65I and S65T,
- wherein said functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein,
- wherein said nucleic acid sequence encoding said fusion protein comprises a polynucleotide sequence encoding said polypeptide of interest operably linked to a polynucleotide sequence encoding said functional mutant fluorescent protein, and
- b) observing a fluorescence characteristic of said at least one cell or said fusion protein or said functional mutant fluorescent protein.
3 more elements
A method for monitoring expression of at least one of a first polynucleotide sequence encoding a first polypeptide of interest or a second polynucleotide sequence encoding a second polypeptide of interest in a cell, comprising the steps of;
- a) providing at least one cell, comprising:
- 1) said first polynucleotide sequence encoding a first fusion protein comprising a first polypeptide of interest and a first functional mutant fluorescent protein, said first functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least one amino acid,
- wherein said first functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein, and
- 2) said second polynucleotide sequence encoding a second fusion protein comprising a second polypeptide of interest and a second functional mutant fluorescent protein, said second functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least one amino acid,
- wherein said second functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of either said Aequorea wild type green fluorescent protein or said first functional mutant fluorescent protein,
- b) observing a fluorescence characteristic of said at least one cell, said first fusion protein said second fusion protein, said first functional mutant fluorescent protein or said second functional mutant fluorescent protein.
5 more elements
A cell, comprising:
- a) a first nucleic acid sequence encoding a first fusion protein comprising a first polypeptide of interest and either 1) an Aequorea wild type green fluorescent protein (SEQ ID NO:2) or 2) a first functional mutant fluorescent protein, said first functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of said Aequorea wild type green fluorescent protein by at least one amino acid, wherein said first functional mutant fluorescent protein has an excitation spectrum different from an excitation spectrum of said Aequorea wild type green fluorescent protein,
- wherein said first nucleic acid sequence encoding said first fusion protein comprises a polynucleotide sequence encoding said first polypeptide of interest operably linked to a polynucleotide sequence encoding said Aequorea wild type green fluorescent protein or said first functional mutant fluorescent protein, and
- b) a second nucleic acid sequence encoding a second fusion protein comprising a second polypeptide of interest and a second functional mutant fluorescent protein, said second functional mutant fluorescent protein comprises:
- an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein by at least one amino acid,
- wherein said second functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of either said Aequorea wild type green fluorescent protein or said first functional fluorescent mutant protein.
3 more elements
A sample, comprising:
- a) at least one first cell comprising a first nucleic acid sequence encoding a either 1) a wild-type fluorescent protein or 2) a first functional mutant fluorescent protein, said first functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of a Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least one amino acid,
- wherein said first functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein,
- wherein said first nucleic acid sequence encoding said wild type fluorescent protein or said first functional mutant fluorescent protein is operably linked to a first promoter, and
- b) at least one second cell comprising a second nucleic acid sequence encoding a second functional mutant fluorescent protein, said second functional mutant fluorescent protein comprising:
- an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO:2) by at least one amino acid,
- wherein said second functional mutant fluorescent protein has an excitation spectrum or emission spectrum different from an excitation spectrum or emission spectrum of either said Aequorea wild type green fluorescent protein or said first functional mutant fluorescent protein,
- wherein said second nucleic acid sequence encoding said second functional mutant fluorescent protein is operably linked to a second promoter.
5 more elements
Dependencies are inferred from the claim text.