Web sites was accomplished using PSORT (Emanuelsson et al., 2007, Nakai Kanehisa, 1991), LipoP
Internet sites was done employing PSORT (Emanuelsson et al., 2007, Nakai Kanehisa, 1991), LipoP (Juncker et al., 2003), and SpLip (Setubal et al., 2006). Protein electrophoresis and immunoblotting Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting have been completed as described previously (Fenno et al., 1996). T. denticola cultures have been harvested in the presence of 2 mM phenylmethylsulfonyl fluoride (PMSF) byAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptMol Oral Microbiol. Author manuscript; obtainable in PMC 2015 September 08.Goetting-Minesky et al.Pagecentrifugation at 10,000 g (10 min, 4 ), washed 1in PBS and suspended in PBS at an optical density of 0.2 at 600 nm, then subjected to Triton X-114 extraction (Cunningham et al., 1988) with no partitioning to aqueous and detergent phases. Equal volumes of samples had been ready in typical SDS-PAGE sample buffer containing mercaptoethanol and 2 mM PMSF. Prior to electrophoresis, samples had been either heated at 100C for five min or held on ice. Proteins blotted to GAS6, Human (HEK293, Fc) nitrocellulose membranes were detected with rabbit polyclonal IL-8/CXCL8 Protein Source antibodies raised against recombinant T. denticola proteins (Fenno et al., 1996, Godovikova et al., 2011b, Godovikova et al., 2010, Bian et al., 2005), followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Thermo Scientific, Rockford, IL). 6xHis-tagged proteins have been detected HisProbe HRP reagent (Thermo Scientific). Protein bands of interest had been visualized employing SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). Protease activity assay PrtP-dependent hydrolysis from the chromogenic substrate succinyl-L-alanyl-L-alanyl-Lprolyl-L-phenylalanine-p-nitroanilide (SAAPFNA) in 5-day T. denticola cultures was assayed by alter in absorbance at 405 nm, as described previously (Uitto et al., 1988, Bian et al., 2005). SAAPFNA activity is expressed as % of SAAPFNA activity of strain 35405 defined as one hundred . Error bars represent the common error over four independent experiments including triplicate samples.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptResults and DiscussionT. denticola TDE0762 (prtP) does not contain an “authentic frameshift” The DNA sequence of T. denticola prtP was initially reported 1996 and assigned Genbank accession D83264 (Ishihara et al., 1996). The T. denticola ATCC 35405 genome was published in 2004 (Seshadri et al., 2004). In addition to the complete genome sequence (Genbank accession NC_002967), two key websites maintain the annotated genome sequence: the Center for Microbial Resources at the J. Craig Venter Institute (cmr.jcvi.org) and also the Oral Pathogen Sequence Database at Los Alamos National Laboratory (Oralgen; oralgen.lanl.gov). While the DNA sequence on the genomic region which includes TDE0762 (prtP) has remained unchanged within the databases, the reported TDE0762 DNA sequence at these websites has varied over the last six years, and TDE0762 (prtP) is annotated as a “pseudogene” (Genbank) or as containing an “authentic frameshift” (CMR, Oralgen). In regular genome annotation methodology, open reading frames are detected using Glimmer (Salzberg et al., 1998). Detection of a mismatch in any specific open reading frame among the genomic sequence along with a previously deposited Genbank sequence results in annotation of your gene as containing an “authentic frameshift” (Nelson et al., 2003). One particular consequence of this convention is the fact that the amino acid seque.