Efficient suppression of the amber codon in E. coli in vitro translation system.
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.
Agafonov
Dmitry E
DE
Laboratorium für Biochemie, Universität Bayreuth, 95440 Bayreuth, Germany.
Huang
Yiwei
Y
Grote
Michael
M
Sprinzl
Mathias
M
D001483:Base Sequence; D018388:Codon, Terminator; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D016153:Genes, Suppressor; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis
D018388:Codon, Terminator; D017931:DNA Primers