Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases.
Takahito Mukai, Takatsugu Kobayashi, Nobumasa Hino, Tatsuo Yanagisawa, Kensaku Sakamoto, Shigeyuki Yokoyama
Index: Biochem. Biophys. Res. Commun. 371(4) , 818-22, (2008)
Full Text: HTML
Abstract
We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA(Pyl) pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA(Pyl) in Chinese hamster ovary cells. When tRNA(Pyl) was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA(Pyl) pair facilitated the most efficient incorporation of a pyrrolysine analog, N(epsilon)-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA(Pyl) system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N(epsilon)-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N(epsilon)-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.
Related Compounds
Related Articles:
Evidence that malondialdehyde-derived aminoenimine is not a fluorescent age pigment.
2001-05-01
[Chem. Res. Toxicol. 14(5) , 473-5, (2001)]
2013-01-07
[Mol. Pharm. 10(1) , 187-98, (2013)]
Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose.
1999-11-15
[Biochem. J. 344 Pt 1 , 109-16, (1999)]
1984-01-01
[Int. J. Pept. Protein Res. 23(1) , 47-54, (1984)]
Well-defined homopolypeptides, copolypeptides, and hybrids of poly(l-proline).
2011-06-13
[Biomacromolecules 12 , 2396-2406, (2011)]