Like an origami crane, a protein must undergo a series of delicate folding steps before settling into the correct shape. For researchers seeking to mass-produce a newly discovered protein, a glow-in-the-dark protein isolated from a jellyfish may help them tell if their target protein has taken the right steps. The technique, described in this month's Nature Biotechnology, may help scientists efficiently determine the structure and function of tens of thousands of unknown proteins found in genome studies.
To produce large enough quantities of a protein for experiments, researchers typically insert the DNA encoding the protein into the bacteria Escherichia coli, turning it into a protein factory. But some proteins overwhelm the bacterium's protein-folding machinery and become an insoluble clump of misfolded molecules. "Instead of correctly folded proteins, you get scrambled eggs," says biochemist Jonathan King of the Massachusetts Institute of Technology. And if the protein's function is unknown or difficult to measure in a test tube, it can be nearly impossible to tell whether or not a protein has folded correctly--that is, as it would have in the organism it came from.
Molecular biologist Geoffrey Waldo of the Los Alamos National Laboratory in New Mexico and his colleagues have found a way around the problem. They attached the gene for a protein called green fluorescent protein (GFP), originally isolated from the Aequorea victoria jellyfish, to the tail ends of genes for 20 relatively unknown proteins that they were trying to produce in E. coli. They found that GFP's brightness under fluorescent light corresponded closely with the percentage of soluble protein. Apparently, when the first protein misfolds, the GFP gets tangled as well and does not take the shape that allows it to glow, the researchers say. When the E. coli protein factories glow bright green--signaling a correctly folded GFP--it is unlikely that the protein has misfolded, Waldo says.
The team also used the technique to identify minor mutations that helped a protein with a strong tendency to misfold in E. coli find its correct shape more easily. They deliberately caused random mutations in the gene and then used their new fluorescence method to identify the protein that folded best. The easy-folding variant was still active.
Quickly identifying such variants will allow scientists to efficiently produce large quantities of correctly folded protein for structural studies, King says. And from a protein's structure, he adds, scientists can make rational guesses about its function.