NIGH LEVEL, CONTEXT-DEPENDENT MISINCORPORATION OF LYSINE FOR ARGININEIN SACCHAROMYCES-CEREVISIAE A1 HOMEODOMAIN EXPRESSED IN ESCHERICHIA-COLI

The Saccharomyces cerevisiae al homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium, Nuclear m agnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass s pectroscopic and NMR analysis demonstrates that the heterogeneity is l argely due to a lysine misincorporation at the arginine (Arg) 115 site . Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E . coli genes, Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorpora tion in al homeodomain production is dependent on media composition, T he dnaY gene, which encodes the rare 5'-ACA-3' tRNA(ARG), was co-expre ssed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression i n minimal media, and homogeneous protein is produced.