coli. To induce gene expression

in the recombinant E. coli, the cells were incubated at 37°C for 2-3 h until the optical density (OD, 600 nm) reached 1.0. Subsequently, 0.1% L-arabinose was added to the culture. During the induction of gene expression, the cell culture was incubated at room temperature (RT) for 16 h. J774A.1 mouse macrophage cells (JCRB9108) were provided by Health Science Research Resources Bank (Osaka, Japan). The J774A.1 cells were cultivated at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum, 100 U penicillin, and 100 μg/ml streptomycin sulfate. Nucleic acid extraction and purification Plasmid and genomic DNA were

extracted according to the method find more described in a previous study [45]. TA cloning, inverse PCR, and Selumetinib in vivo DNA sequencing A fragment of pnxIIIA was amplified with the primer pair pnx2A-f and pnx2A-r by using Ex Taq (Takara Bio, Shiga, Japan), and the amplified product was purified using SUPREC-PCR (Takara Selleckchem Adriamycin Bio). The purified PCR amplicons were ligated with the pTAC-1 vector (Biodynamics Laboratory, Tokyo, Japan), and E. coli DH5α was transformed with the resultant vectors. The clones were screened via blue-white selection and direct colony PCR by using the M13 primer pair. For inverse PCR, the genomic DNA of P. pneumotropica ATCC 35149 was digested with various restriction enzymes that recognized a 6-nucleotide sequence, and subsequently, the digestion product was self-ligated with T4 ligase (Takara Bio) and then used as an inverse PCR template. Inverse PCR was performed using gradient PCR to determine the optimum annealing temperature for a model DNA Engine PTC-200 (Bio-Rad Laboratories, Hercules, CA, USA). The PCR products were ligated with the pTAC-1 vector and screened to ensure the accuracy of sequencing. Cycle sequencing was performed using the BigDye terminator premix Cyclin-dependent kinase 3 (Applied Biosystems, Foster City, CA, USA). The products of the sequencing reaction were analyzed using an ABI 310 or ABI 3730XL DNA analyzer (Applied

Biosystems). Purification of recombinant Pnx proteins rPnxIIIA was extracted and purified from the cell culture of E. coli strain TMU0812 harboring pBAD-Pnx3A. The cultured cells were suspended in 20 mM Tris-HCl, 150 mM NaCl, 5 mM imidazole, and 1 mM 2-mercaptoethanol (pH 8.0, binding buffer); they were then broken by sonication. The sonicate was centrifuged at 7,000 × g for 10 min and filtered using a 0.45-μm filter unit (Millipore, Billerica, MA, USA). The supernatant was loaded onto a 1-ml His-trap HP affinity column (GE Healthcare, Amersham, UK) mounted on an ÁKTAprime plus fast protein liquid chromatography device (FPLC device; GE Healthcare), and chromatography was performed by running a program for histidine-tagged protein purification according to the manufacturer’s instructions.