Characterization of New and Improved Fluorescent Proteins and their Applications

Abstract

Fluorescent proteins (FPs) have become ubiquitous tools in biological and biomedical research. Since the cloning and exogenous expression of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, researchers have sought new variants of this and other FPs, with properties well-suited for particular imaging applications. Of special interest are FPs with different excitation and emission wavelengths, with brighter fluorescence, monomeric, and that mature rapidly at 37°C. We have examined the properties of several novel FPs isolated from fluorescent marine organisms, which were collected from the Great Barrier Reef in Australia and Belizean Barrier Reefs. All the proteins were cloned and expressed in E. coli and their properties were characterized. One FP, 28bc2, showed very promising characteristics having high brightness and a weak propensity to form dimers. We have modeled the structure of 28bc2 and designed mutations at the putative dimerization interface to decrease the potential to dimerize. We have further characterized the spectroscopic properties of 28bc2 wild type (wt) and the mutants and compared their properties with those of EGFP, a widely used variant of GFP. The 28bc2 wt and mutants FP are at least 2-fold brighter than EGFP and show similar pH stability profiles to that of EGFP. The photostability of 28bc2 wt and mutants is less than that of EGFP, though for some applications this is not critical. We have shown the advantages of brightness of 28bc2 mutants in one-step detection application in Western blotting and their usefulness in in vivo labeling demonstrated by RNA microinjection assays in zebrafish.