期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 371, 期 4, 页码 818-822出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2008.04.164
关键词
pyrrolysine tRNA; acetyllysine; benzyloxycarbonyllysine; non-natural amino acid; protein engineering
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 (PyIRS)-tRNA(PYl) pair from Methanosarcino 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 PyIRS-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 PyIRS 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. (c) 2008 Elsevier Inc. All rights reserved.
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