Fig. 7

Western blot and tandem mass spectrometry (MS/MS) analyses of His-tagged recombinant PEP-CTERM protein of PepA in the A. tertiaricarbonis strains. The samples were taken at 12 h, 18 h and 24 h. Immunoblotting was performed with His-specific monoclonal mouse antibody followed by a goat anti-mouse IgG conjugated to HRP. A PVDF membrane incubated with His-tag-specific antibody Western ECL (Upper panel) and three bands, upper, middle and lower, appeared; a PVDF membrane incubated with anti-RpoA antibody (Generated using the RpoA recombinant protein of closely related Zoogloea resiniphila MMB strain as the antigen) Western ECL as a loading control (Middle panel) and also a Coomassie Brilliant Blue stain of the SDS-PAGE gel as a loading control (Lower panel). Lane M: The size marker; Lane1-Lane 3: The proteins extracted from the RN12T4 mutant carrying the empty vector pBBR1MCS-2 over the time course; Lane 4-Lane 6: The proteins extracted from RN12T4 mutant carrying pBBR1MCS-2-pepA over the time course. The molecular weight of the marker bands is indicated in kilo-Daltons (KD). Mass spectrometry identification of the upper (B), middle band (C) and lower band (D) detected by the anti-His tag antibody in the Western blots. The MS/MS sequence coverage of proteins is 31, 37 and 30%, respectively. MS/MS spectra of the tryptic peptide APHHHHHHVFEVTPSVLGAPGSPFNANK (m/z = 756.129) confirmed the presence of the hexahistindine tag in all three bands. The western blots used in figures were from two regions of one membrane (The RpoA was about 35KD and the PepA was lower than 29KD)