The 29-and 27-kDa proteins were mainly detected in the cytoplasm/periplasm fraction of the wild type and hbp35 insertion mutant (Figure 2). Figure 2 Subcellular localization of HBP35. Subcellular fractions of P. gingivalis 33277 (lanes 1 to 5) and KDP164 (hbp35 insertion mutant) (lanes 6 to 10) were subjected to immunoblot analysis using anti-HBP35 antibody. MK2206 Lanes 1 and 6, whole cells; lanes 2 and 7, cytoplasm/periplasm fraction; lanes 3 and 8, total membrane fraction; lanes 4 and 9, inner membrane fraction; lanes 5 and 10,

outer membrane fraction. Horizontal lines between lane 5 and 6 indicate the molecular size marker proteins corresponding to the far left markers. Asterisks indicate the non-specific protein bands recognized by anti-HBP35 antibody. Peptide Mass Fingerprint analysis of the 27-kDa protein To determine whether the 27-kDa protein is a truncated form of the HBP35 protein, an immunoprecipitation experiment using the hbp35 insertion mutant (KDP164) cell lysate was carried out with the anti-HBP35 antibody.

The resulting immunoprecipitate contained a 27-kDa protein band (Additional file 2), which was digested with trypsin followed by MALDI-TOF mass spectrometric analysis. The 27-kDa protein was found to be derived from a 3′-portion of hbp35, with PMF sequence coverage of 37% of full length protein (Figure 3A). The maximum mass error among the identified 10 tryptic peptides was 14 ppm. Since the detected tryptic peptide located at the most N-terminal region of HBP35 starts from G137 and since Daporinad the insertion site of the ermF-ermAM DNA cassette in the insertion mutant is just upstream of F110, it is feasible that the 27-kDa protein uses M115 or M135 as the alternative translation initiation site. Figure 3 Identification of the anti-HBP35-immunoreactive 27-kDa protein and the start codons of anti-HBP35-immunoreactive proteins. A. PMF

analysis of the anti-HBP35-immunoreactive 27-kDa protein from KDP164 (hbp35 insertion mutant). Underlined peptide fragments were indicated by the PMF data of the protein. Bold letters indicating M115 and M135 were suspected to be internal start codons. B. Bumetanide Immunoblot analysis of P. gingivalis mutants with various amino acid substitutions of HBP35 protein. Lane 1, KDP164 (hbp35 insertion mutant); lane 2, KDP168 (hbp35 [M115A] insertion mutant); lane 3, KDP169 (hbp35 [M135A] insertion mutant); lane 4, KDP170 (hbp35 [M115A M135A] insertion mutant). Identification of the N-terminal amino acid residue of truncated HBP35 proteins To clarify the N-terminal amino acid residue of the truncated HBP35 proteins, we introduced amino acid substitution mutations of [M115A] or/and [M135A] to the hbp35 insertion mutant (KDP164) producing the 29-and 27-kDa HBP35 proteins (Additional file 3).