Increasing Unnatural Amino Acid Incorporation 5-Fold in Mammalian Cells
Inventors: Jeffrey K. Takimoto, Katrina L. Adams, Zheng Xiang and Lei Wang
Potential Uses: Bioproduction, Fermentation, Gene Expression, Research Tool
Improved orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells
Unnatural amino acids (UAAs) are a recent powerful tool that enables novel chemical and physical properties to be selectively introduced into proteins directly in live cells, thus having great potential for addressing molecular and cell biological questions in the native cell settings. These materials also allow specific, directed modifications of protein for bioproduction and medical use. To date, UAAs have been genetically encoded in E. coli, yeast, and mammalian cells using orthogonal tRNA-synthetase pairs and unique codons.
The incorporation efficiency of UAAs is currently low in mammalian cells, compared to E. coli and yeast. Inefficient incorporation results in reduced yield of proteins containing the unnatural amino acid, and in turn its use for research investigations and bioproduction. In this invention, we were able to increase the efficiency up to 5-fold. To do this we optimized the anticodon recognition between the orthogonal tRNA and the tRNA synthetase, demonstrating its use in a variety of applications, including cross-linking of proteins in living cells. This method can expand the use of UAAs in biological research and bioproduction with mammalian cells.
Patent Status: U.S. Patent Application filed July 2009
Publications: Jeffrey K. Takimoto, Katrina L. Adams, Zheng Xiang and Lei Wang Mol. BioSyst., 2009, 5, 931 - 934. Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells
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