کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1935197 | 1050660 | 2008 | 5 صفحه PDF | دانلود رایگان |
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)-tRNAPyl pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNAPyl in Chinese hamster ovary cells. When tRNAPyl was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNAPyl pair facilitated the most efficient incorporation of a pyrrolysine analog, Nε-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNAPyl 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 Nε-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to Nε-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.
Journal: Biochemical and Biophysical Research Communications - Volume 371, Issue 4, 11 July 2008, Pages 818–822