Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase.
We describe the heterologous expression of a recombinant Saccharomyces cerevisiae isoleucyl-tRNA synthetase (IRS) gene in Escherichia coli, as well as the purification and characterization of the recombinant gene product. High level expression of the yeast isoleucyl-tRNA synthetase gene was facilitated by site-specific mutagenesis. The putative ribosome-binding site of the yeast IRS gene was made to be the consensus of many highly expressed genes of E. coli. Mutagenesis simultaneously created a unique BclI restriction site such that the gene coding region could be conveniently subcloned as a "cassette." The variant gene was cloned into the expression vector pKK223-3 (Brosius, J., and Holy, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6929-6933) thereby creating the plasmid pKR4 in which yeast IRS expression is under the control of the isopropyl-thio-beta-galactopyranoside (IPTG)-inducible tac promoter. Recombinant yeast IRS, on the order of 10 mg/liter of cell culture, was purified from pKR4-infected and IPTG-induced E. coli strain TG2. Yeast IRS was purified to homogeneity by a combination of anion-exchange and hydroxyapatite gel chromatography. Inhibition of yeast IRS activity by the antibiotic pseudomonic acid A was tested. The yeast IRS enzyme was found to be 10(4) times less sensitive to inhibition by pseudomonic acid A (Ki = 1.5 x 10(-5) M) than the E. coli enzyme. E. coli strain TG2 infected with pKR4, and induced with IPTG, had a plating efficiency of 100% at inhibitor concentrations in excess of 25 micrograms/ml. At the same concentration of pseudomonic acid A, E. coli strain TG2 infected with pKK223-3 had a plating efficiency less than 1%. The ability of yeast IRS to rescue E. coli from pseudomonic acid A suggests that the eukaryotic synthetase has full activity in its prokaryotic host and has specificity for E. coli tRNA(ile).
Racher
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Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Kalmar
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Borgford
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TJ
D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D007533:Isoleucine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016712:Mupirocin; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D004271:DNA, Fungal; D005227:Fatty Acids; D016712:Mupirocin; D007533:Isoleucine-tRNA Ligase