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Efficient suppression of the amber codon in E. coli in vitro translation system
Author(s) -
Agafonov Dmitry E.,
Huang Yiwei,
Grote Michael,
Sprinzl Mathias
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.03.004
Subject(s) - transfer rna , translation (biology) , in vitro , serine , stop codon , release factor , protein biosynthesis , messenger rna , esterase , chemistry , suppressor , microbiology and biotechnology , enzyme , biochemistry , biology , gene , rna
An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNA Ser(CUA) was monitored by determination of the full‐length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild‐type protein.