Construct for high-level expression and low misincorporation of lysine for arginine during expression of pET-encoded eukaryotic proteins in Escherichia coli.
The arginine codon AGA is rarely used in E. coli but is common in eukaryotic genes. Prior studies have shown that the low level of tRNA(UCUArg) can lead to low expression and misincorporation of lysine for arginine, during expression of genes containing AGA codons in E. coli. The chloramphenicol-selectable plasmid pJY2 is designed to facilitate the expression of such genes cloned into pET vectors: it encodes T7 lysozyme (to depress constitutive expression of the cloned gene) and tRNA(UCUArg) (to suppress lysine misincorporation at AGA codons). Using pJY2, we observed robust and translationally faithful expression of mutant ubiquitin genes in which 14% (11 out of 76) of the total codons were AGA. Competent BL21(DE3)pJY2 cells can be used to suppress lysine misincorporation and achieve high-level expression of pET-encoded target genes without modification of AGA codons in the target gene sequence.
You
J
J
Johns Hopkins University, Baltimore, MD, USA.
Cohen
R E
RE
Pickart
C M
CM
D001120:Arginine / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001704:Biopolymers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D008239:Lysine / Q000235:genetics; D013058:Mass Spectrometry; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D025821:Polyubiquitin; D014176:Protein Biosynthesis; D012347:RNA, Transfer, Arg / Q000235:genetics; D011994:Recombinant Proteins; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D001704:Biopolymers; D012347:RNA, Transfer, Arg; D011994:Recombinant Proteins; D014452:Ubiquitins; D025821:Polyubiquitin; D001120:Arginine; D008239:Lysine