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 ε - 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 ε -benzyloxycarbonyl- l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N ε -acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.
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