The DNA sequence of the protease was obtained by a search of the Pseudomonas Genome Project database (www.pseudomonas.com, GenBank Accession #AE004091) using the N-terminal amino acid sequence of the purified protease. PCR primers were constructed to amplify the gene with EcoRI and BamHI restriction sites flanking 5′ and 3′ ends, respectively. The forward primer was 5′-CGGAATTCCATGCTGAAGAAGACCCTT-3′ and the reverse primer was 5′-GGATCCTTACTGGCGAATGCCTTC-3′. The gene was amplified using P. aeruginosa strain PA103 genomic DNA as the template, 50 picomoles of each primer, 2 mM MgCl2, Taq polymerase (Promega, Madison, WI), 10× buffer, 10 mM dithiothreitol, 5% glycerol, 0.01% BSA, and 0.2 mM each of dATP, dCTP, dGTP, and dTTP. The PCR reaction was performed as follows: 100°C for 5 minutes; add Taq polymerase, 94°C for 1 minute; then 30 cycles of 94°C for 20 seconds; 55°C for 20 seconds; and 68°C for 1 minute.
The 573-bp PCR product was purified from a 1% agarose gel with a gel-extraction kit (Qiagen, Valencia, CA). The purified product was ligated into vector pCR2.1 and transformed into E. coli TOP10F′ cells, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Transformants were analyzed by restriction digestion of purified plasmid DNA.
Plasmid pHAT10 (BD-Clontech) was chosen as the expression vector. Plasmids pCR2.1 harboring the cloned protease gene and pHAT10 were digested with BamHI and EcoRI. The digested protease gene and pHAT10 were gel-purified using the gel-extraction kit. The protease gene was ligated into pHAT10, resulting in plasmid pHAT10-Pasp, and transformed into chemically competent E. coli DH5αPRO cells. Colonies that grew on 50 μg/mL ampicillin were chosen for plasmid DNA analysis. Sequencing of the Pasp gene in pHAT10 was performed by the LSUHSC Genomics Core Facility (Louisiana State University Health Sciences Center, New Orleans, LA). The sequencing primer was 5′-AGGCTTTACACTTTATGCTTCCGGCTCGTA-3′, located 189 nucleotides upstream of Pasp in pHAT10.