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The Quest for New Fluorescent Proteins

Nicholas Miliaras, Ph.D.

The biotech community has been trying to improve on the original Green Fluorescent Protein (GFP) for nearly 20 years, but there appears to be no better innovator when it comes to protein engineering than nature herself. Will a newly discovered fluorescent protein replace GFP? Find out...

Since James Remington’s group first published the crystal structure of the original Aequora victoria GFP in 1996, researchers have engineered more than 250 variants of the protein. Early modifications significantly improved fluorescence intensities and biophysical properties, but recently developed fluorescent proteins appear to have reached an upper limit in terms of brightness.

Amphioxus, displaying its fluorescent proteins. Credit: Dimitri Deheyn
Most advances in brightness have come through modification of three key GFP residues, but Dimitri Deheyn, associate researcher at the Scripps Institution of Oceanography at University of California, San Diego, needed a different approach if he planned to burst through the brightness ceiling.

Rather than continuing to mutate those 3 residues, Deheyn and his colleagues turned to other marine organisms, examining 16 different GFPs naturally co-expressed in the cephalochordate Amphioxus. “This is a rare opportunity since most organisms only have one, or maybe a couple of FPs,” he explained. “We were looking to screen as many invertebrates as possible for [brighter] FPs, and when we saw one of the Amphioxus GFPs, it was so much brighter.”

They next compared the photonic, biophysical, and structural properties of a very bright (bfloGFPa1) and very dim (bfloGFPc1) Amphioxus GFP to Aequora eGFP. While they found that the bfloGFPs share similar architectures, excitation spectra, and emission spectra with eGFP, some critical structural differences in the molecular environment around 3 residues of the bfloGFPa1 chromophore made it 3.5× brighter than eGFP and almost 1000× brighter than bfloGFPc1.

Rather than adopting bfloGFPa1 for biological experiments, Deheyn expects that the research community will engineer similar changes to the Aequora GFP chromophore, since it is so well established. In fact, he has already had several requests for details of the Aequora GFP substitutions. According to Deheyn, brighter GFPs should be available very soon as long as there are no unforeseen problems, such as solubility, that arise when incorporating the changes.


Erin K. Bomati, Joy E. Haley, Joseph P. Noel, and Dimitri D. Deheyn. “Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus” Scientific Reports 4: 5469, 2014