A group of researchers at the Massachusetts Institute of Technology have come up with a new way of labeling proteins using a molecule that is small enough so that it does not disrupt the cellular processes of the tagged proteins.
Their invention means researchers have an option not to use the popular but bulky Green Fluorescent Protein. Large fluorescent tags, such as the 238-amino acid GFP, are important in a wide variety of studies, but sometimes the bulk of the protein can interfere with the processes that researchers are trying to observe, scientists have noted.
“In the example of the EGF receptor, putting GFP on it messed up trafficking and caused severe retention in the endoplasmic reticulum,” said Alice Ting, the leader of the research group that developed the new labeling technology, which is described in this week’s issue of Nature Methods. “Because of its size, GFP can interfere with protein processes and functioning.”
In response to this problem, Ting, an assistant professor at MIT’s department of chemistry, and her graduate student, Irwin Chen, engineered a 15-amino acid tag that covalently binds to a ketone when in the presence of a bacterial protein called BirA. The ketone analog is a modified derivative of biotin that can bind any hydrozide molecule, including fluorescent molecules or photo affinity probes, which are good for studying protein-protein interactions.
Virtually any cell-surface protein containing the special acceptor peptide tag can be labeled using the BirA reaction, which takes about 20 minutes, the researchers said in their paper. They demonstrated that tagging a cell-surface receptor with the acceptor peptide and ketone did not interfere with the function of the protein.
“There’s lots of labeling methods out there, but none that are as small and as specific as this one,” Ting told ProteoMonitor this week.
Ting said a patent for the new labeling technology is pending, and that she is currently in discussions with various companies that have expressed interest in commercializing it.
Geoff Waldo, the director of protein engineering resources at Los Alamos Laboratories, said Ting’s new technique has “tremendous” promise. However, one downside, which Ting’s group has acknowledged, is that the method currently only works on cell surfaces. The method does not work in the interior of cells because cells contain small molecules that have ketone functionality. Those molecules would compete with binding to the acceptor peptide tag-bound ketone.
“If they could engineer a biotin ligase to accept a new substrate that is not present in cells, they might be able to adapt the method so that it works within living cells,” said Waldo. “It’s certainly worth putting the effort into. In the future, if they could engineer this to work in living cells, this would be the method of choice for site-specific labeling of proteins.”
The closest technology to Ting’s new ketone-labeling technology that is currently available is Roger Tsien’s FlAsH technology, which has been commercialized by Invitrogen, said Waldo. Tsien is professor of chemical biology at the University of California San Diego.
Ting’s technology has advantages over Tsien’s technology in that the labeling is covalent, and therefore much more stable, and the ligase protein is more specific in labeling than FlAsH, Waldo noted.
“Once you’ve labeled a protein with Ting’s technology, it’s stably labeled forever. You can do whatever you want with it — unfold it, refold it,” said Waldo. “FlAsH is not nearly as stable, and you have to use a bunch of chemical cocktails to try to limit non-specific binding.”
The biggest hurdle in developing the new labeling technique was finding the initial assay to attach biotin to the acceptor peptide, said Chen. The technique took about two years to develop.
From there, it was also challenging to engineer the biotin-derivative ketone, which is the platform for attaching fluorescent tag molecules, Chen added.
“We had to prove that the BirA enzyme would accept the peptide as a substrate,” said Chen. “Once that was found, we had a pretty clear idea of what to do. Getting it to work on a living cell is always challenging as well.”
Chen said that his next project will be to try to adapt the ketone technology so that it works inside cells.
“We’re trying to incorporate a more selective functional group, like an azide,” said Chen.
The biggest challenge of using azides as functional groups will be to find an enzyme that can attach the azide to the acceptor peptide tag protein, said Chen.
Waldo said that one exciting use of Ting’s new technology would be for labeling antibodies.
“The labeling of the antibody could be done outside a cell, and then it could be imported into cells using protein transfection,” said Chen. “Then you can do protein interaction assays.”
Garry Nolan, the director of the National Heart, Lung, and Blood Institute Proteomics Center at Stanford, said Ting’s new technology is a powerful approach for rapidly and genetically labeling surface proteins. He noted that aside from using the approach for cell-surface proteins, the new technology could also be used to label intracellular targets on fixed cells, or in tissue slices.
Aside from Ting and Chen, other researchers who contributed to the development of the new technology were postdoctoral associates Mark Howarth and Weiying Lin.
“This is one of those chemical problems that has been a bottleneck for a lot of biological applications,” said Ting. “We hope that [this new technique] will open up this bottleneck. We want lots of people to use this method.”