HIGH-LEVEL MISINCORPORATION OF LYSINE FOR ARGININE AT AGA CODONS IN AFUSION PROTEIN EXPRESSED IN ESCHERICHIA-COLI

The expression of eukaryotic genes in Escherichia coli is one of the m ost frequently used tools of modern science. The arginine codon AGA is a common codon in eukaryotic genes but is particularly rare in E. col i. We report here 36 to 42% misincorporation of lysine at three AGA co dons in a well-expressed protein. This misincorporation yields a prote in 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 intensi ty, respectively. Replacement of either all three AGA codons or the tw o closest to the 3' end of the gene by the more common CGC arginine co don gave a protein with a single ESMS peak. Misincorporation could als o be eliminated by the co-expression of the tRNA(UCU)(Arg) gene, argU. These studies demonstrate that misincorporation of amino acids at rar e codons of recombinant proteins can be far higher than previously tho ught. (C) 1996 Academic Press Limited