Fluorescent GFP variant displaying highly increased fluorescence intensity without a spectral shift

摘要

The present invention relates to a nucleic acid molecule encoding a polypeptide having a fluorescence emission activity with a maximum emission at 505 to 515 nm, wherein said nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) a nucleic acid molecule having the DNA sequence of SEQ ID NO: 1; (c) a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of (i.) a nucleic acid molecule of (a), wherein said nucleic acid molecule of (c) encodes a polypeptide having at the position corresponding to position 146 of SEQ NO:2 a phenylalanine and at the position corresponding to position 203 of SEQ NO:2 a threonine; or (ii) a nucleic acid molecule of (b), wherein said nucleic acid molecule of (c) has at the positions corresponding to positions 438 to 440 of SEQ ID NO: 1 a nucleotide triplet selected from the group consisting of TTT and TTC; and at the positions corresponding to positions 609 to 611 of SEQ ID NO: 1 a nucleotide triplet selected from the group consisting of ACT, ACC, ACA, ACG; wherein the polypeptide encoded by the nucleic acid molecule of (c) has a fluorescence enhanced by at least the factor of 2.5 as compared to the polypeptide having the amino acid sequence of SEQ ID NO: 10; or (d) a nucleic acid molecule degenerate with respect to the nucleic acid molecule of (b). The present invention furthermore relates to a polypeptide encoded by the nucleic acid molecule of the invention, a vector and a host cell comprising the nucleic acid molecule of the invention, a method of producing said polypeptide, a fusion protein comprising the polypeptide of the invention and methods of detecting the presence and/or localization of a protein of interest and methods of detecting the activity of a promoter.

主权项

1. An isolated or purified nucleic acid molecule encoding a polypeptide having a fluorescence emission activity with a maximum emission at 505 to 515 nm, wherein said nucleic acid molecule is selected from the group consisting of
(a) a nucleic acid encoding a polypeptide SEQ ID NO: 2; (b) a nucleic acid having the DNA sequence of SEQ ID NO: 1; and (c) a nucleic acid that hybridizes under stringent conditions to the complementary strand of
the nucleic acid of (a), and wherein said nucleic acid of (c) encodes a polypeptide having a phenylalanine at position 146 of SEQ ID NO:2 and a threonine at position 203 of SEQ NO:2; and (d) a nucleic acid that hybridizes under stringent conditions to the complementary strand of the nucleic acid of (b), and wherein said nucleic acid of (d) has at positions 438 to 440 of SEQ ID NO: 1 a nucleotide triplet selected from the group consisting of TTT and TTC; and at positions 609 to 611 of SEQ ID NO: 1 a nucleotide triplet selected from the group consisting of ACT, ACC, ACA, ACG; and wherein the nucleic acid molecule encodes a polypeptide that has a fluorescence emission activity with a maximum emission at 505 to 515 nm and has a fluorescence enhanced by at least the factor of 2.5as compared to the polypeptide having the amino acid sequence of SEQ ID NO: 10.