Cell-permeable fluorescent proteins
This invention relates to methods and compositions for designing novel fluorescent proteins, preferably to a green fluorescent proteins (GFP). The engineered GFPs are modified by substituting negatively charged amino acids with positively charged amino acids on the exterior of the protein making the protein cell permeable. The ability of the engineered fluorescent proteins to permeate cells obviates the need for transfections, allowing these novel proteins to be used in numerous biological applications.
1. An engineered fluorescent protein comprising:
a) a fluorescent protein differing in amino acid sequence from the reference protein of (i) SEQ ID NO: 1-3 or 5, wherein the difference consists of substituting a glutamic acid or an aspartic acid with an arginine or a lysine at positions 17, 19, and 21 of SEQ ID NO:1-3 or 5, or (ii) SEQ ID NO: 4, wherein the difference consists of substituting a glutamic acid or an aspartic acid with an arginine or a lysine at positions 18, 20, and 22 of SEQ ID NO: 4, wherein the modified protein is permeable to a cell;
b) a peptide linker region linked at either the amino or carboxyl end of the modified fluorescent protein, wherein the linker region is a polypeptide between about 1 and 30 amino acid residues in length and is susceptible to digestion by a cellular protease; and
c) a fluorescence modifier moiety linked to the other end of the linker region, wherein a fluorescence resonance energy transfer occurs between the modified fluorescent protein and the fluorescence modified moiety in the uncleaved engineered fluorescent protein, and wherein when the protease cleaves the linker region of the engineered fluorescent protein in a cell, a change in fluorescence occurs in the cell as compared to a reference cell that contains the uncleaved engineered fluorescent protein, but not the cellular protease.
2. The engineered protein as claimed in claim 1 wherein the linker has the amino acid sequence TSFNFPQITC (SEQ ID NO: 13) and the protease is human immunodeficiency virus type 1 (HIV-1) protease.
3. The engineered protein as claimed in claim 1 , wherein the fluorescence modifying moiety is a tetramethyl rhodamine.
4. An engineered fluorescent protein for detecting a protease activity in a cell, the engineered protein comprising:
a) a fluorescent protein differing in amino acid sequence from the reference protein of (i) SEQ ID NO: 1-3 or 5, wherein the difference consists of substituting a glutamic acid or an aspartic acid with an arginine or a lysine at positions 17, 19, and 21 of SEQ ID NO:1-3 or 5, or (ii) SEQ ID NO: 4, wherein the difference consists of substituting a glutamic acid or an aspartic acid with an arginine or a lysine at positions 18, 20, and 22 of SEQ ID NO: 4, where the modified protein is permeable to a cell;
b) a peptide linker region linked at either the amino or carboxyl end of the modified fluorescent protein, wherein the linker region is a polypeptide between about 1 and 30 amino acid residues in length and is susceptible to digestion by a cellular protease; and c) a fluorescence modifier moiety linked to the other end of the linker region, wherein a fluorescence resonance energy transfer occurs between the modified fluorescent protein and the fluorescence modified moiety in the uncleaved engineered fluorescent protein, and wherein when the protease cleaves the linker region of the engineered fluorescent protein in a cell, a change in fluorescence occurs in the cell as compared to a reference cell that contains the uncleaved engineered fluorescent protein, but not the cellular protease,
wherein the change in fluorescence indicates the protease activity in the cell.
5. The engineered protein as claimed in claim 4 , wherein the linker has the amino acid sequence TSFNFPQITC (SEQ ID NO: 13) and the protease is human immunodeficiency virus type 1 (HIV-1) protease.
6. The engineered protein as claimed in claim 4 , wherein the fluorescence modifying moiety is a tetramethyl rhodamine.
7. An assay method for detecting activity of a protease in a cell, the method comprising:
exposing the engineered protein of claim 4 to at least one experimental cell;
measuring fluorescence emission intensity of the cell to obtain a measured fluorescence intensity; and
comparing the measured fluorescence intensity of the experimental cell with the fluorescence intensity of a reference cell that has the engineered protein, but not the protease, wherein a difference in the fluorescence intensity between the two cells is indicative of the presence of the protease in the experimental cell.
8. The method as claimed in claim 7 , wherein the at least one cell is from cells grown in culture or a cell sample from a mammalian host.
9. The method as claimed in claim 7 , wherein the protease is human immunodeficiency virus type 1 (HIV-1) protease.
10. A kit for assaying cells for activity of a protease, the kit comprising any one of the engineered fluorescent proteins as claimed in claim 4 .
11. The kit as claimed in claim 10 further comprising instructions for use.
12. An assay method for detecting activity of a protease in a cell, the method comprising:
a) exposing an engineered fluorescent protein to at least one experimental cell, wherein the protein comprises:
i) a fluorescent protein differing in amino acid sequence from the reference protein of SEQ ID NO: 1-3 or 5, wherein the difference consists of substituting a glutamic acid or an aspartic acid with an arginine or a lysine at positions 17, 19, and 21 of SEQ ID NO:1-3 or 5, or SEQ ID NO: 4, wherein the difference consists of substituting a glutamic acid or an aspartic acid with an arginine or a lysine at positions 18, 20, and 22 of SEQ ID NO: 4, where the modified protein is permeable to a cell;
ii) a peptide linker region linked at either the amino or carboxyl end of the modified fluorescent protein, wherein the linker region is a polypeptide between about 1 and 30 amino acid residues in length and is susceptible to digestion by a cellular protease; and
iii) a fluorescence modifier moiety linked to the other end of the linker region, wherein a fluorescence resonance energy transfer occurs between the modified fluorescent protein and the fluorescence modified moiety in the uncleaved engineered fluorescent protein, and wherein when the protease cleaves the linker region of the engineered fluorescent protein in a cell, a change in fluorescence occurs in the cell as compared to a reference cell that contains the uncleaved engineered fluorescent protein, but not the cellular protease;
b) measuring fluorescence emission intensity of the cell to obtain a measured fluorescence intensity; and
c) comparing the measured fluorescence intensity of the experimental cell with the fluorescence intensity of a reference cell that has the engineered protein, but not the protease, wherein a difference in the fluorescence intensity between the two cells is indicative of the presence of the protease in the experimental cell.