Methods of Genetically Encoding Unnatural Amino Acids in Eukaryotic Cells Using Orthogonal TRNA/Synthetase Pairs
Inventors: Lei Wang
Potential Uses: Research Tool, Protein Production
Genetic encoding of novel amino acids in mammalian cells, high-yield production of unnatural amino acids and novel proteins.
Proteins participate in various biological processes and can be harnessed to probe and control biological events selectively and reproducibly. However, the genetic code limits protein building blocks to 20 common amino acids, which limits our ability to manipulate proteins in live cells. The ability to genetically encode unnatural amino acids would remove this restriction and enable novel chemical and physical properties to be precisely introduced into cellular proteins. Historically, genetically encoding unnatural amino acids in mammalian cells has been greatly hampered by two main challenges: inefficient biosynthesis of orthogonal bacterial tRNAs and difficulty in generating unnatural-amino-acid-specific synthetases in these cells. Consequently, this type of engineering has been mainly restricted to bacteria or in yeast, and it has been difficult to incorporate unnatural amino acids in mammalian cells.
Salk researchers have developed a novel strategy to expand the natural repertoire of 20 amino acids in mammalian cells and have successfully inserted tailor-made amino acids into proteins in these cells. The method involves "coercing" mammalian cells to express bacterial tRNAs by using the H1 promoter, then pairing the tRNAs with synthetases that recognize the tRNAs and attach the cognate unnatural amino acid. In proof-of-principle experiments, unnatural amino acids with extended side chains were used to determine the operating mechanism of the ?molecular gates? that regulate the movement of potassium ions in and out of nerve cells, a finding that eluded conventional amino acid mutagenesis.
Genetically encoding unnatural amino acids will allow more precise investigation of biological events directly in living cells and organisms. Site-directed mutagenesis using novel amino acids will become possible, allowing large-scale production of mutant proteins useful for sample-intensive in-vitro studies, as well as for therapeutic and industrial applications. Other possible applications include unnatural amino acids tailor-designed and encoded to probe, and even control, proteins and protein-related biological processes. For example, fluorescent unnatural amino acids could be used to sense local environmental changes and serve as reporters for enzyme activity, membrane potential or neurotransmitter release. Those bearing photocrosslinking agents could be applied to identify protein-protein and protein-nucleic acid interactions in cells; and photoisomerizable amino acids could be designed to switch on and off signal initiation and transduction noninvasively.
Patent Status: U.S. Patent Application published as 2008/0254540
International Patent Application published as WO 2008/127900 A1
Publications: J Am Chem Soc. 130(19): 6066-7 (2008)
Nature Neuroscience 10(8): 1063-1072 (2007)
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