Don’t feel blue; a brighter fluorescent protein is now here
Blue fluorescent proteins have been created from GFP, but they tend to photobleach too rapidly and to fluoresce insufficiently for cellular imaging or screening applications. Now a new variant of those proteins -- called Azurite -- is significantly brighter and remains fluorescent much longer after excitation than the fluorophore from which it was created.
Azurite was genetically engineered from a blue fluorescent protein by researchers at the University of California, Santa Barbara, and at California Institute of Technology in Pasadena. They created a library of E. coli expressing hundreds of thousands of blue fluorescent protein variants. Because they believed that molecular movements within the fluorophore reduced its brightness, they made targeted mutations in the core of the protein that they hypothesized would stabilize it. They determined the mutations that would theoretically yield brighter fluorophores by using a computational algorithm for library design.
To find those E. coli expressing fluorescent proteins with the best photophysical properties, the investigators screened the library by fluorescence-activated cell sorting using a BD Biosciences flow cytometer. They monitored the fluorescence from the best candidates with a fluorometer from Tecan Group Ltd.
Azurite fluoresced 60 percent more brightly than did standard blue fluorescent protein, owing to a quantum yield of 0.55 for Azurite versus 0.34 for the original.
Fluorescence measurements of Azurite performed via time-correlated single-photon counting with a PI/Acton monochromator and a Hamamatsu microchannel plate photomultiplier tube revealed a fluorescence lifetime of 3.65 ns, whereas normal blue fluorescent protein has a lifetime of only 2.21 ns.
Finally, the researchers examined Azurite in mammalian cells with the Tecan fluorometer. The fluorophore fluoresced 40 times longer than conventional blue protein. In both bacterial and mammalian cells, it exhibited less pH sensitivity than traditional blue fluorescent protein. The scientists reported these findings in the December 2006 issue of Nature Biotechnology.
Because Azurite is 60 percent brighter, 40 times more photostable and less pH-sensitive than is standard blue fluorescent protein, the researchers concluded that it will work much better than previous blue proteins for all fluorescence applications, including multicolor cytometry, fluorometry and microscopy.
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