GFP - Imaging Fluorescent Proteins

Introduction

The green fluorescent protein (wt GFP) from the jellyfish Aequorea Victoria (1) is widely used in fluorescence microscopy. Although the wild type form has undergone many mutations to optimize its properties as a tool for biological investigations, a process that is still going on. One of those mutants is the commercially distributed EGFP which has a red-shifted excitation maximum and is brighter than the wtGFP. To reach higher expression levels its cDNA codon usage has been optimized for the mammalian translation system. Further mutations of GFP led to the generation of a colorful group of fluorescent proteins (referred to as XFPs) and there is a continuous effort to develop new improved variants.

Most commercially available XFPs can be ordered from BD Biosciences (formerly Clontech).

XFPs used today — overview (there are more!)

_{order: increasing excitation wavelength}

	Ex-Max (nm)	Em-Max (nm)	Comments
BFP			low quantum yield (dim), strong photobleaching
CFP			bright, very little photobleaching but: long maturing time
ECFP	439	476
EGFP	484	510	bright, very little photobleaching
YFP (1st gen.)			high sensitivity towards pH and Cl- photobleaching stronger than EGFP
YFP (2nd gen.)	512	529	less pH sensitive than 1st gen. YFP
3rd gen. YFPs (3, 4)			no Cl- - sensitivity, little pH-sensitivity, less photobleaching, faster maturation
EYFP	512	529
RFP (DsRed)	563	582	tetrameric, wide excitation shoulder, long maturing time, partly folded protein fluoresces green, long emission _: good for tissue labelling
Fluorescent Timer (DsRed variant, Clontech)			uses DsRed characteristic: during folding color changes from green to red to observe promoter activity over time
T1 (DsRed Express, Clontech)			maturing time only 10 min
mRFP1 (5)	500/584	607	new monomeric RFP

For more information on fluorecent proteins, please refer to:

• Overview of spectral properties of XFPs.
• Fluorescence filter information for XFPs from Chroma.

A second group of fluorescent proteins is derived from reef corals and has consequently been named Reef Coral Fluorescent Proteins (RCFPs). Although deriving form a different class of organism, RCPs share structural homology with GFP. Unlike the color variants of Aequora GFP, which represent mutant variants of a single fluorescent protein, RCPs are unique proteins encoded by distinct genes. They are not generally recommended for use as protein tags. DsRed (from the list above) is one representative of this group. BD-Biosciences offers 6 spectrally distinct proteins from the family.

Recent developments

Photoactivatable YFP-mutant: PA-GFP (6)
undergoes a 100-fold increase in fluorescence at 488nm when illuminated before at 413nm

Color-changing protein Kaede (7)
converts from a green (475nm exc.) to a red (550nm exc.) fluorescent protein after irradiation with 350-400nm light

Application for both: localized labeling of organelles or protein subpopulation which can then be followed in space and time (protein trafficking, turnover, etc.)

Destabilized XFPs (BD Bioscience/Clontech)
EGFP with half-lives of 1-4 hours

Applications

• measure kinetics of transient mRNA transcription from regulated promoters
• monitor gene expression during development
• characterize cis-regulatory elements