Mutant blue fluorescent protein and method of using the same for fluorescence resonance energy transfer and blue fluorescent fish

Abstract

The present invention discloses a mutant blue fluorescent protein (BFP) exhibiting, at an aerobic or anaerobic system, larger fluorescent intensity than a BFPvv D7 of SEQ ID NO:2 derived from a wild type BFP, BfgV of SEQ ID NO:1, obtained from Vibrio vulnificus, wherein a set of mutation positions of the mutant BFP corresponding to SEQ ID NO:2 comprises position 176 or position 178. In a preferred embodiment, the set of mutation positions of the mutant BFP corresponding to SEQ ID NO:2 comprises a S176R mutation or a V178I mutation. Moreover, methods of using the blue fluorescent proteins from Vibrio vulnificus for fluorescence resonance energy transfer (FRET) and a blue fluorescent fish are also provided.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to the field of molecular and cellular biology. More specifically, the present invention relates to a mutant blue fluorescent protein (BFP) capable of existing in an anaerobic or aerobic system and methods of using the same for fluorescence resonance energy transfer (FRET) and a blue fluorescent fish.[0003]2. Description of Related Art[0004]Fluorescent proteins, such as green fluorescent proteins (GFPs) from Aequorea victoria or GFP-like fluorescent proteins, have become an invaluable tool in cell biology. Over the last several years, GFP variants with altered fluorescence spectra, improved folding properties, increased brightness and altered pH-sensitivity have been increased (Tsien, 1998; Chudakov et al., 2005), and these GFP variants are widely used in the fields of biochemistry, molecular and cell biology, medical diagnostics and drug screening methodologies.[0005]When GFP family protei...

AI Technical Summary

Benefits of technology

[0010]Accordingly, an objective of the present invention is to provide a novel mutant blue fluorescent protein (BFP) having improved fluorescent properties comprising higher fluorescence quantum yield (QY), stronger fluorescence intensity and not sensitive to photobleaching at not only high temperature condition but at an anaerobic environment.

[0015]In brief, a mutant BFP and methods of using the same for FRET and a fluorescent fish in accordance with the present invention provide one or more of the following advantages:

[0017](2) The mutant BFP of the present invention has improvements in both brightness and photostability, thereby suitable to apply for FRET, particularly for multiple-FRET imaging with single-wavelength excitation, which is a powerful method for detection of protein-protein interaction, enzyme activities and small molecules in the intracellular milieu, so as to be able to report biochemical phenomena in living cells. Additionally, it is also suitable to apply for producing a blue fluorescent fish.

[0019](4) The mutant BFP of the present invention will not lose its luminance even in anaerobic conditions, not like all GFP-like fluorescent proteins. As different from GFP-like proteins, a NADPH-dependent mutant BFP will illuminate through NADPH as a chromophore; being put in more simple terms, the oxygen-dependent maturation process won't be needed in the mutant BFP of the present invention. In addition, UV-excitation (352 nm) permits the mutant BFP to illuminate visible blue light (440 nm) in vivo without oxygen.

[0022](7) The mutant BFP of the present invention may be applied for a BiFC-FRET assay. The basic rationale of BiFC-FRET assay is combination of both BiFC and FRET assays to visualize ternary complexes in living cells. In the BiFC-FRET assay, two proteins (A and B) are being blended to two nonfluorescent fragments obtained from fluorescent protein GFP, but the third protein (C) is fused to the full-length mutant BFP of the present invention. The interaction between proteins A and B will then be further recompose an intact GFP, and it will then serves as a FRET acceptor. Should protein C interact with proteins A or B or both, the interaction can bring the mutant BFP (FRET donor) close to the reconstituted GFP and it allows FRET to occur. Hence, BiFC-FRET assay can work to provide evidence of ternary complex formation.

Problems solved by technology

Although a few enhanced BFPs (EBFPs) have been developed by introducing several mutations into BFPs, EBFPs are rarely used so far because of still having a undesirably low fluorescence quantum yield (QY), thereby being weakly fluorescent, and remaining relatively sensitive to photobleaching (Kremers et al., 2007).

In addition, one major drawback shared by most newly discovered wild-type fluorescent proteins is that they are dimeric.

Heterodimerization is undesirable when fluorescent proteins are used to be expressed as a fusion to another protein of interest or when they are used in FRET.

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