Cloning and Purification of P. aeruginosa LeuRS

The gene encoding P. aeruginosa LeuRS was obtained through PCR amplification (MJ Mini Thermo Cycler, Bio­Rad, Hercules, CA) using P. aeruginosa PAO1 (ATCC 47085) genomic DNA as a substrate. A forward primer (5’-gaacgctagccacgagcaatatcagccact-3’), designed to add an Nhel restriction site to the 5’ end of the gene, and a reverse primer (5’-ggataagctttcagttggcgacgatgttg −3’), which was designed to add a HindIII restriction site to the 3’ end of the gene, were used in PCR. The PCR product was inserted into a pET-28b(+) plasmid (Novagen) digested with NheI/HindIII. This resulted in the gene, encoding P. aeruginosa LeuRS, fused to an upstream region encoding six histidine residues. The recombinant plasmid was transformed into E. coli Rosetta 2(DE3) Singles Competent Cells (EMD Millipore, Danvers, MA). Bacterial cultures were grown, expression of the target protein induced and P. aeruginosa LeuRS was purified as described [10 (link)].