After three rounds of selection (positive, negative, positive), the growth rates of 96 clones were assayed individually on selective media in the presence or absence of 1. Three different clones were isolated that are dependent on 1 for growth. The ability of these clones to incorporate 1 into proteins selectively was tested by suppression of an amber mutant (Trp-33 TAG) of His₆-tagged human superoxide dismutase (hSOD-33TAG-His₆), and subsequent SDS/PAGE analysis with either GelCode Blue (Pierce) staining or fluorescence imaging. LeuRS mutant B8 in which all five active site residues were mutated (M40A, L41N, Y499I, Y527G, and H537T) afforded the most protein in the presence of 1; however, significant amounts of hSOD-33TAG-His₆ were also produced in the absence of 1. Analysis of hSOD-33TAG-His₆ expressed in the presence of 1 by MALDI-TOF MS indicated that leucine or isoleucine was also being incorporated at position 33.

Redesign of the Editing Domain and Enhancement of tRNA Expression. E. coli LeuRS has an editing site to enhance selectivity toward structurally similar amino acids, e.g., isoleucine, valine, and methionine. Recent structural and biochemical studies have shown that hydrolysis of a cognate activated or charged leucine at this site is prevented by steric repulsion between one or more active-site amino acids and a -methyl group of leucine. Mutation of one such residue, Thr-252, to alanine (T252A) results in efficient hydrolysis of aminoacylated tRNA^Leu (23, 24). Consequently, to enhance fidelity of LeuRSB8, the T252A mutation was introduced (LeuRSB8T252A). Analysis of the suppression efficiency with hSOD-33TAG-His₆ revealed relatively low yields of protein in the presence of 1 [0.29 mg/liter hSOD-33TAG-His₆ harboring 1 after nickel-nitrilotriacetic acid (Ni-NTA) purification]. However, the T252A mutation resulted in a marked reduction of hSOD expression in the absence of 1. Previous studies of the suppression efficiency of an amber suppressor E. coli tRNA^Tyr/TyrRS pair in yeast indicated that suppressor tRNA levels can limit overall protein yields (S.C., unpublished results). To increase tRNA expression, three copies of tRNA_CUA^Leu5 with flanking regions from yeast suppressor tRNA SUP4, which are known to contain an RNA polymerase (pol) III promoter, were inserted into the plasmid pLeuRSB8T252A (25, 26). These multiple tRNA genes were placed under the control of an additional phosphoglycerate kinase 1 (PGK1) promoter, resulting in an 4-fold enhancement (1.11 mg/liter) of expression of hSOD harboring 1 at position 33 (Fig. 2A). A high degree of fidelity for incorporation of 1 was confirmed by MALDI-TOF MS {calculated mass, m/z, 16,802; observed mass, 16,802 ([M+H]⁺); Fig. 2B}. No peaks corresponding to incorporation of endogenous amino acids were observed. Moreover, no protein was observed in the SDS/polyacrylamide gel in the absence of 1 (Fig. 2A).

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