, Osaka, Japan), Ala-Phe-4-nitroanilide (Bachem AG, Bubendorf, Switzerland), and Ala-Phe-Pro-4-nitroanilide (Bachem AG), respectively, as substrates. The reactions were performed at 37 °C, and A405 nm was measured with a SPECTRA max 384 plus (Molecular Devices, Sunnyvale, CA). For verification of inner membrane fractions, the NADH–ferricyanide oxidoreductase activity was determined by measuring A420 nm in 80 mM Tris-HCl pH 7.4, 9.0 mM KCN, 1.0 mM NADH, and 0.7 mM ferricyanide (Futai, 1974). Lipopolysaccharide was isolated using the lipopolysaccharide Extraction Kit (Intron Biotechnology Inc., Kyunggi, Korea) according to the manufacturer’s protocol, separated
by SDS-PAGE, and visualized using the nondiamine silver staining method (Merril, 1990), with a slight modification. Gels were thoroughly fixed with methanol (10%)–acetic acid (5%). Then, gels were fixed in methanol (50%)–formaldehyde (0.02%) for 20 min, soaked in dithiothreitol (0.03 mM) for 20 min, stained with http://www.selleckchem.com/products/AZD6244.html AgNO3 (0.1%) for 20 min, and rinsed with deionized water three times.
Image development was achieved in Na2CO3 (3%)–formaldehyde (0.02%), and was stopped Nutlin-3a manufacturer in 3% acetic acid. pTYXB-His (Ishiguro et al., 2009) was digested with NcoI and EcoRI, and ligated with an annealed-oligonucleotide linker [5′-CATGCTGCAGTGAATTCCATCACCATCACCATCACT-3′/5′-AATTAGTGATGGTGATGGTGATGGAATTCACTGCAG-3′ (italics: PstI and EcoRI sites)] to generate pTYPE-His, which carries PstI and EcoRI cloning sites and the sequence for a histidine tag. For the expression of the PG534 antigen, the PstI–EcoRI-digested 1.3-kbp PG0534 fragment from pKS39 was ligated to the PstI–EcoRI-digested pTYPE-His, generating pKS45, which encodes 404Q–827F of PG534 with a C-terminal histidine tag (-His-His-His-His-His-His). The PG0694 gene encodes an OmpA homologue PG694 (Nagano et al., 2005). For the expression of the PG694 antigen, the PG0694 gene was amplified by PCR using 5′-CACTGCAGGAAGCTACTACACAGAACAAAGCAGGG-3′ (italics: PstI site) and 5′-CGAATTCCATTACAGGGAAGTCTGCTTTTCCTCTC-3′ (italics: EcoRI site), digested Dapagliflozin with PstI and EcoRI, and ligated to the PstI–EcoRI-digested pTYPE-His,
generating pKS46, which encodes 22Q-212M of PG694 with a C-terminal histidine tag (-Glu-Phe-His-His-His-His-His-His). ER2566(pKS45) and ER2566(pKS46) were grown in Luria–Bertani broth supplemented with isopropyl-β-d-thiogalactopyranoside (0.3–0.5 mM). The recombinant protein was purified from inclusion bodies using Ni2+-chelated Sepharose Fast Flow (GE Healthcare UK Ltd., Buckinghamshire, UK) under denaturing conditions, according to the manufacturer’s protocol. Purified proteins were dialyzed against distilled water and then injected into a rabbit to prepare antiserum. The antisera were designated anti-PG534 and anti-PG694. PG0534 encodes a putative protein, PG534, 837 amino acids in length (Nelson et al., 2003). We constructed three Porphyromonas gingivalis mutants for the PG0534 gene: 83K8, 83K25, and 83K26 (Fig. 1a).