This Week's Citation Classic: GFP

This week's citation classic is M Chalfie, Y Tu, G Euskirchen, WW Ward, and DC Prasher.
Green fluorescent protein as a marker for gene expression. Science 11 February 1994:
Vol. 263. no. 5148, pp. 802 - 805. DOI: 10.1126/science.8303295.

Martin Chalfie has just been awarded a Nobel Prize for his work relating to the development of one biology's most ubiquitous tools: green fluorescent protein. In one of Chalfie's first experiments, he marked six individual C. elegans cells in the with the aid of GFP.

GFP allows "the monitoring in time and space of an ever-increasing number
of phenomena in living cells and organisms like gene expression, protein localization and
dynamics, protein-protein interactions, cell division, chromosome replication and organization,intracellular transport pathways, organelle inheritance and biogenesis, to name but a few" [Nobelprize.org].

Chalfie first found out about GFP in 1988 during a seminar on bioluminescent organisms by Paul Brehm. Brehm's seminar reported that GFP, a protein found in the jellyfish, Aequoria victoria, fluoresces green in the presence of blue light.

Chalfie thought to himself, "What an absolutely wonderful, wonderful compound this would be. I would love to put it into C. elegans and look at gene expression. In fact I imagined a really large number of things I would like to do if I had such a way of marking gene expression and protein localization in living cells. I got very excited and didn’t listen to another word of the seminar."

Chalfie contacted Doug Prasher at Woods Hole who was attempting to clone the GFP cDNA. Chalfie and Prasher agreed to get in touch when Prasher succeeded with the cloning, but due to missed connections, four years passed before Chalfie saw the published sequence in Gene.

Chalfie contacted Prasher and asked "What happened to our collaboration? Do you still want to do this?". Prasher was unable to get the GFP to fluoresce but sent the protein to Chalfie anyway. Chalfie then gave it to his grad student Ghia Euschirken. Two weeks later, Euschirken had something to show Chalfie: green fluorescent E. coli.

Chalfie describes why he was able to get GFP to fluoresce while others failed:

I was very concerned that we only use the coding sequence and no flanking DNA in our construct. We used PCR to obtain just the coding sequence and that worked terrifically. But the others who tried to use GFP simply cut the original lambda cDNA clone with restriction enzymes, and got additional bases associated with each end. These extra sequences turned out to be quite detrimental; expression wouldn’t occur. So we were fortunate in the way we designed the experiment.

Chalfie and Euschirken eventually cloned GFP into worms as a promoter driven construct, and the rest is history.

On a sad note, Prasher no longer works in science. He is now driving a courtesy shuttle for a car dealership in Huntsville, Ala.

An interview with Chalfie is available here.