Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases.
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.
Mukai
Takahito
T
Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Kobayashi
Takatsugu
T
Hino
Nobumasa
N
Yanagisawa
Tatsuo
T
Sakamoto
Kensaku
K
Yokoyama
Shigeyuki
S
D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005815:Genetic Code; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000235:genetics* / Q000378:metabolism; D008250:Lysine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D017020:Methanosarcina / Q000201:enzymology; D014176:Protein Biosynthesis; D015202:Protein Engineering
D019843:Archaeal Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; C040741:epsilon-tert-butyloxycarbonyl-lysine; D008250:Lysine-tRNA Ligase; C456839:pyrrolysine; D008239:Lysine; C417029:EBV-encoded nuclear antigen 1