Scientists at North Carolina State University and the Medical Research Council in Cambridge, UK, have devised a new method for protein labeling.
The technique, which enables researchers to genetically encode fluorophore-binding amino acids, offers faster, more efficient tagging than previous labeling approaches and could prove useful in a range of applications, including cell surface protein labeling and live cell imaging, Alexander Deiters, associate professor of chemistry at NC State and, with MRC researcher Jason Chin, one of the developers of the method, told ProteoMonitor.
In a study published last week in the online edition of Nature Chemistry, Deiters and his colleagues used the technique to perform site-specific protein labeling in E. coli and mammalian HEK293 cells.
The method uses pyrrolysyle-tRNA synthetase/tRNACUA pairs to direct the site-specific encoding of norbornene-containing lysines into proteins of interest. This norbornene can then be made to bind with tetrazine-based probes that exhibit fluorescence upon their reaction with the encoded norbornene group.
The approach, Deiters said, was enabled by developments in recent years in the use of tRNA synthetase/tRNACUA pairs.
These reagents have allowed researchers to genetically encode a range of bioorthogonal groups like azides and ketones that can be used in protein labeling. The slow reactivity of such previously explored bioorthogonal functional groups has limited their utility, however, he said.
For instance, if researchers wanted to label a protein to track its movement through a cell, existing reactions might prove too slow for them to obtain an accurate picture of its location.
"The sluggishness of established bioorthogonal reactions often makes it challenging to label proteins quantitatively at defined sites in vitro," the authors wrote, noting that this "may account for the fact that there are currently no examples of labeling proteins expressed on the mammalian cell surface using genetically encoded unnatural amino acids."
The norbornene-tetrazine reaction, on the other hand, proceeds quickly enough to make such applications possible. "If you want to, say, do live cell imaging, you can use this technology to quickly label it with a fluorophore and then see how it translocates to another compartment, or similar experiments," Deiters said.
In the Nature Chemistry study, he and his colleagues used the technique to successfully label epidermal growth factor receptor on the surface of HEK293 cells. They also attempted to label EGFR via an azide-alkyne reaction using a TAMRA-DIBO-alkyne probe from Invitrogen, but were unable to observe specific labeling with this approach.
"The [azide] technology and this technology were tested side-by-side, and basically with the azide no labeling in live mammalian cells was observed, while with our technology very nice labeling was observed," Deiters said. "This, I think, is the first example where bioconjugation of fluorophores was conducted on the surface of live mammalian cells."
According to Deiters the norbornene approach offers a binding reaction that is roughly 50 times faster than previous methods as well as a higher yield of labeled proteins. In addition to enabling improved labeling, the faster binding time reduces the opportunity for the reaction to interfere "with the normal function of the proteins and cells being studied," he said.
Using the pyrrolysyle-tRNA synthetase/tRNACUA pairs, researchers should be able to place norbornene-containing amino acids anywhere within a protein, Deiters said, although he noted that it is necessary to make sure the insertion site is accessible by the labeling fluorophore in the folded protein.
"It's a fairly easy process," he said, noting that his team succeeded in its attempts to insert unnatural amino acids into different proteins nine out of 10 times.
Moving forward, Deiters said, the researchers plan to extend the technique to bioconjugating other types of entities, "for example, it would be interesting to use this to add types of polymers to proteins or to immobilize a protein on a surface."
"I think, as we showed [in the paper], protein labeling in live cells is one excellent application" of the technique, he said. "Labeling proteins with biophysical probes would also be an interesting application — spin labels, for example. And you could also use it to make therapeutic proteins – bioconjugating small-molecule cytotoxic agents to proteins and things like that."
The study, Deiters said, was not aimed at any specific biological question but was, rather, intended as "pure technology development.
"We looked at what was going on in terms of bioconjugation reactions and realized that a lot of development has been done, but that they are still fairly slow and not super efficient," he said. "So, we thought we would develop this new approach. Protein bioconjugations are important tools and we wanted to extend that tool box by [developing] a faster and more efficient site-specific protein bioconjugation."
Deiters said the researchers have filed patents covering the technology, and while he is not intimately involved in pursuing commercialization of it, if the group found "partners interested in picking this up … then we'd certainly be interested in commercializing it."
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