High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli.
The expression of eukaryotic genes in Escherichia coli is one of the most frequently used tools of modern science. The arginine codon AGA is a common codon in eukaryotic genes but is particularly rare in E. coli. We report here 36 to 42% misincorporation of lysine at three AGA codons in a well-expressed protein. This misincorporation yields a protein whose electrospray mass spectrum (ESMS) shows peaks at the expected mass (M), M-28, M-56 and M-84 with intensities representing 34.5(+/-0.7), 37.5(+/-1.1), 21.2(+/-1.7) and 6.6(+/-0.5) % of the total intensity, respectively. Replacement of either all three AGA codons or the two closest to the 3' end of the gene by the more common CGC arginine codon gave a protein with a single ESMS peak. Misincorporation could also be eliminated by the co-expression of the tRNA(UCL)Arg gene, argU. These studies demonstrate that misincorporation of amino acids at rare codons of recombinant proteins can be far higher than previously thought.
Calderone
T L
TL
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Stevens
R D
RD
Oas
T G
TG
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*
D003062:Codon; D011993:Recombinant Fusion Proteins; D001120:Arginine; D008239:Lysine