Optimising Blue Fluorescent Protein (BFP) for use as a mammalian reporter gene in parallel with Green Fluorescent Protein (GFP).

Etienne Joly

doi:10.1038/npre.2007.1259.1

Abstract

AbstractTo obtain an optimised form of BFP for use as a reporter gene in mammalian cells, the brightest available GFP form, EGFP, was mutated at 5 different positions, yielding 8 different mutagenised forms of BFP. The intensity of the fluorescent signals attained in mammalian cells with all these various versions of BFP was analysed by flow cytometry of transiently transfected COS 7 cells. The best mutant obtained can be detected readily both by flow cytometry and fluorescence microscopy, even when expressed together with GFP. To explore whether cellular localization could enhance the fluorescence signals any further, plasmid constructs were made to target optimised versions of GFP and BFP to the nucleus, the endoplasmic reticulum (ER) and the cell surface. Expression in the nucleus or ER increased the fluorescence signal by ca. 50%, whereas cell surface expression resulted in a five-fold decrease compared to the ER and nuclear forms. Co-expression of GFP and BFP in the same cellular compartment did not result in any significant absorption of the blue fluorescence by GFP. Thus, targeting of GFP and BFP to various cellular compartments adds even further versatility to this convenient dual reporter-gene system.

Highlights

Over the past few years, Aequorea victoria 's green fluorescent protein (GFP) has proven an invaluable tool to study gene expression, protein localization and sub-cellular compartment dynamics
The fluorescence signals obtained with BFP and GFP are separated, but two major drawbacks of BFP are that its fluorescence is much weaker than that of GFP and the UV required for excitation results in rapid bleaching in fluorescence microscopy
To obtain an optimised version of BFP to use in parallel with GFP in mammalian cells, a commercially available version of GFP called EGFP (Clontech) was chosen

Summary

Introduction

Over the past few years, Aequorea victoria 's green fluorescent protein (GFP) has proven an invaluable tool to study gene expression, protein localization and sub-cellular compartment dynamics. Of the variants reported so far, the most useful are derived from the Y66H mutation which leads to emission of blue fluorescence [1,2,3]. The overlap of the green and yellow spectra, make this variant unsuitable for analysis by fluorescence microscopy in parallel with GFP, and separate detection by flow cytometry is possible only with carefully controlled electronic compensation. BFP has not been very widely used in mammalian cells as a reporter gene mainly because of these drawbacks. Efficient protein expression can be affected if the nucleic acid sequence shows a bias towards codons that are rarely used by the organism, as was demonstrated by Haas et al on expression of HIV-1 glycoprotein, Thy-1 and GFP in human cells [5]. Several mutations resulting in an increase of the protein's stability at 37°C have been reported by different groups [9,10,11,12]

Results

Discussion

Conclusion