J. Mol. Biol. (1996) 262, 407–412 High-level Misincorporation of Lysine for Arginine at AGA Codons in a Fusion Protein Expressed in Escherichia coli Tiffany L. Calderone 1 , Robert D. Stevens 2 and Terrence G. Oas 1 * The expression of eukaryotic genes in Escherichia coli is one of the most 1 Department of Biochemistry and 2 Mass Spectrometry 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 Facility, Division of Medical Genetics, Department of report here 36 to 42% misincorporation of lysine at three AGA codons in Pediatrics, Duke University a well-expressed protein. This misincorporation yields a protein whose Medical Center, Durham 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), NC 27710, USA 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 Arg UCU gene, argU. These studies demonstrate that misincorporation of amino acids at rare codons of recombinant proteins can be far higher than previously thought.  1996 Academic Press Limited Keywords: mistranslation; low-usage codons; AGA; tRNA Arg UCU *Corresponding author It is important to monitor translation errors in studies using overexpressed recombinant eukary- otic and viral genes. The misincorporation of amino acids can drastically affect protein function and lead to a discrepancy between apparent and actual specific activity (Schimmel, 1989). The arginine codon AGA is a common eukaryotic codon but is extremely rare in Escherichia coli genes (de Boer & Kastelein, 1986; Zhang et al ., 1991). For this reason, when eukaryotic genes are expressed in E. coli without prior modification of codon usage, the occurrence of AGA codons is quite likely. The effect of these low-usage codons on the sequence of the expressed protein is often overlooked. Translation errors during protein synthesis are a well-documented phenomenon (Parker, 1989). They can have several different effects including frame shifts (Spanjaard et al ., 1990), premature truncation, low expression (Brinkmann et al ., 1989; Rosenberg et al ., 1993) and misincorporation (Seetharam et al ., 1988; Schimmel, 1989). The majority of previously reported translation errors have fallen into the first three categories. This has led to the general view that codon usage in recombinant proteins is important primarily for determining expression levels, particularly in the first ten to 20 codons (Parker, 1989; Spanjaard et al ., 1990; Rosenberg et al ., 1993). If expression levels are sufficient and the protein shows the correct apparent molecular mass on an SDS-PAGE gel, translational integrity is often assumed to be intact. This assumption is not always valid, as demon- strated here. Although low levels of misincorporation at several low-usage codons have previously been detected in recombinant proteins, the levels are generally low enough that the mistranslated protein is only a minor contaminant (Parker, 1989). It has been proposed that adjacent AGA codons are necessary to cause translation errors (Rosenberg et al ., 1993). In the only published report of misincorporation above 1%, the expressed protein purposely contained six AGA codons leading to 12% misincorporation at two adjacent AGA codons (Seetharam et al ., 1988). We have observed considerably higher misincorporation (36 to 42%) at three non-adjacent AGA codons and have shown that the misincorporation is due to low levels of the corresponding tRNA Arg UCU molecule. Abbreviations used: ESMS, electrospray mass spectrum; IPTG, isopropyl--D-thiogalactopyranoside; CNBr, cyanogen bromide. 0022–2836/96/390407–06 $18.00/0  1996 Academic Press Limited