Enhanced D-amino acid incorporation into protein by modified ribosomes

By overexpression of modified Escherichia coli 23S rRNAs from multicopy plasmids, ribosomes were prepared that contained mutations in two regions (2447-2450 and 2457-2462) of 23S rRNA. Following mutagenesis and selection, two clones with mutations in the 2447-2450 region (peptidyltransferase center) and six with mutations in the 2457-2462 region (helix 89) were characterized. The mutations were shown to exhibit a high level of homology. Cell-free protein synthesizing systems prepared from these clones were found to exhibit significantly enhanced incorporation of d-methionine and d-phenylalanine into protein. The incorporations involved positions 10, 22, and 54 of E. coli dihydrofolate reductase and positions 247 and 250 of Photinus pyralis firefly luciferase. Interestingly, some of the derived proteins containing the d-amino acids (notably DHFR analogues altered at position 10) functioned as well as those containing the respective l-amino acids, while substitution at other positions resulted in proteins having greatly diminished activity.