In our previous study [1], high levels of leucine aminopeptidase (LAP) enzymatic activity had been detected in both clinical and environmental isolates of B. pseudomallei, by APIZYM analysis (bioMérieux, Marcy l’Etoile, France). LAP which belongs to the peptidase M17 family, is involved in the processing and regular turnover of intracellular proteins by catalyzing the removal of unsubstituted N-terminal amino acids from various peptides [3, 4]. Besides proteolytic Selleck AZD3965 activities, this enzyme is also known to play an important role as a DNA-binding protein in Escherichia coli[5], and a repressor or activator in

the operon regulation of virulence-associated genes in E. coli, Vibrio cholerae and Pseudomonas aeruginosa[6–8]. The LAP enzyme has been proposed as an immunoantigen for vaccination

against Fasciola hepatica in sheep [9, 10] and a promising drug target for Helicobacter pylori infections [11]. As there has not been any study on LAP of B. pseudomallei, the objective of the present study was to characterise the LAP activity of B. pseudomallei and to examine the intra- and inter-species variation in the nucleotide and deduced amino acid sequences of the LAP encoding gene (pepA). A pepA/PCR-RFLP was designed to facilitate the identification of LAP sequence types and for possible differentiation of phenotypically identical B. pseudomallei isolates. Methods Extraction of Inhibitor Library LAP One milliliter of an overnight-culture of B. pseudomallei NCTC 13178 (McFarland 3) was inoculated into 3 liters of BHI broth and incubated at 37°C for 72 h with constant agitation at 120 rpm in a shaker (DAIKI SCIENCES Co., Ltd., Korea). The bacterial cells were removed by centrifugation at 4,500 rpm for 30 min at 4°C, and the flow-through filtered using a 0.2 μm polyethersulfone membrane (Sartorius Stedium Biotech, Germany). One part of the filtrate was mixed with 2 parts of cold saturated ammonium sulfate solution for 10 min with stirring, prior to centrifugation at 12,000 rpm for 45 min

at 4°C. The precipitate was dissolved in cold 50 mM Tris-HCl buffer (pH 7.6). Desalting was performed using HiPrep 26/10 desalting column (GE Healthcare Bio-Sciences, Sweden) Silibinin coupled to a AKTA™ explorer 100 system (GE Healthcare Bio-Sciences, Sweden). The eluent was concentrated using a Vivaspin 15R column (MWCO 5,000 molecular cut-off, Sartorius Stedium Biotech, Germany) by centrifugation at 6,000 g. The protein concentration of the sample was determined by Quick Start™ Bradford Protein Assay (Bio-Rad, US) using bovine serum albumin as the standard. Zymographic analysis Zymographic analysis was performed to detect the presence of LAP activity in the crude extract of B. pseudomallei NCTC 13178. The extract was diluted 40 fold (0.64 mg/ml) and mixed with NativePAGE™ buffer (4 X) (Invitrogen Corporation, Carlsbad) in a ratio of 3:1.