Another novel tagging and detection system has been developed by Geoffrey Waldo, Ph.D., team leader, biosciences, Los Alamos National Laboratories. “Existing small tags usually require a labeled antibody or the like,” he says. “These can be hard to use especially in living cells. We are trying to overcome this by making families of tiny tags and a specific detector protein for each tag. The detectors bind like antibodies to each particular tag.
“The trick is they can be expressed right in the cell and give a fluorescent signal only when they bind a tag. We are engineering several different fluorescent proteins from various organisms. We split them into two unequal pieces. One is a tiny piece of about 15 amino acids that acts as the tag to attach to the protein. The larger remaining fragment acts as the detector. Neither piece is fluorescent alone. By carefully engineering the properties of the fragments, the larger detector fragment acts like an antibody to the tag and it spontaneously binds to become brightly fluorescent, as bright as the original full-length fluorescent protein.”
Dr. Waldo is applying the technology to protein trafficking, protein interaction detection, high-throughput measurement of soluble protein in living cells, and engineering proteins for stability and solubility.
Aminoacyl-tRNA synthetases within cells serve to ligate a specific amino acid with its cognate tRNA. The tRNA subsequently contributes that amino acid to the growing peptide chain. Researchers at Princeton University are harnessing that power to incorporate unnatural amino acids as novel tags in recombinant proteins.
“In the last decade a lot of effort has been focused on engineering aminoacyl-tRNA synthetases to incorporate unnatural amino acids into recombinant proteins,” reports A. James Link, Ph.D., assistant professor, chemical engineering and molecular biology. “This is useful for a number of applications. For example, a fluorescent molecule can be linked that will allow tracking the protein intracellularly. For therapeutic applications a polymer such as polyethylene glycol could be added to allow the protein to remain in circulation. Such tags can functionalize a protein. We found a new way to introduce this functionality into proteins.”
Functional groups such as azides and alkynes have emerged as chemical handles to help couple tags to proteins. Dr. Link found that an efficient way to introduce such functional groups is via incorporation of unnatural amino acids.
“Previously we used combinatorial screening techniques to identify new aminoacyl-tRNA synthetases. We found a variant of the E. coli methionyl-tRNA synthetase (MetRS) with the capability to incorporate either its natural amino acid or the unnatural amino acid azidonorleucine into a protein depending on which amino acid it is presented with. In our latest work, we generated an E.coli strain that harbors a single genomic copy of this engineered MetRS.”
Because the bacterial strain can ligate either the unnatural amino acid or its natural substrate (methionine), these strains can be considered dual-purpose organisms. According to Dr. Link, “the genetic code changes as a function of whether or not the unnatural amino acid or the natural methionine is present in the medium in which the E. coli grows. We first grow the cell in the presence of methionine (since it cannot grow with the unnatural amino acid), then we remove that and switch the medium to one containing the azidonorleucine. We harvest and purify the protein and often get yields as high as 20–30 mg protein per liter.”
Once azidonorleucine has been introduced into a protein, the click chemistry reaction (a reaction between azide and alkyne functional groups) can be used to tag the protein with a wide variety of tags including biotin, fluorophores, polymers, and even conjugated metals for imaging applications.
Dr. Link is now working to utilize the bacterial strain in producing uniquely substituted proteins and as a tool for modeling host-pathogen interactions.