The gene encoding P. aeruginosa MetRS was obtained through PCR amplification (MJ Mini Thermo Cycler, Bio-Rad, Hercules, CA) using P. aeruginosa PAO1 (ATCC 47085) genomic DNA as a substrate. A forward primer (5′-ATATGCTAGCTCCGAACCACGCAAGATC-3′), designed to add an NheI restriction site to the 5′ end of the gene and the reverse primer (5′-CTCTAAGCTTTTACTTGACGCGCTGGC-3′) which was designed to add a HindIII restriction site to the 3′ end of the gene, were used in the PCR. The PCR product was inserted into a pET-28b(+) plasmid (Novagen) digested with NheI/HindIII. The recombinant plasmid was transformed into E. coli Rosetta 2(DE3) Singles Competent Cells (EMD Millipore, Danvers, MA).
The E. coli bacterial cultures were grown in Terrific Broth containing 25 μg/mL of kanamycin and 50 μg/mL of chloramphenicol at a temperature of 37 °C to an optical density (A600) of 0.6–0.8. The over-expression of P. aeruginosa MetRS in the cultures was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 0.25 mM. Growth of the culture was continued for 2 hours post-induction, and cells were harvested using centrifugation (10,000 g, 30 min, 4 °C). Fraction I lysates were prepared as previously described [7 (link)]. P. aeruginosa MetRS was purified to more than 98% homogeneity as previously described [6 (link)].
The E. coli bacterial cultures were grown in Terrific Broth containing 25 μg/mL of kanamycin and 50 μg/mL of chloramphenicol at a temperature of 37 °C to an optical density (A600) of 0.6–0.8. The over-expression of P. aeruginosa MetRS in the cultures was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 0.25 mM. Growth of the culture was continued for 2 hours post-induction, and cells were harvested using centrifugation (10,000 g, 30 min, 4 °C). Fraction I lysates were prepared as previously described [7 (link)]. P. aeruginosa MetRS was purified to more than 98% homogeneity as previously described [6 (link)].
