Purification of RgpA from external outer membrane vesicles of Porphyromonas gingivalis

Abstract

IntroductionPurification of native gingipains is challenging because these proteases are frequently associated with the cell surface, which affects yield. This study aimed to purify native Arg-gingipain (RgpA) from Porphyromonas gingivalis Outer Membrane Vesicles (OMV). MethodsNative RgpA was purified from P. gingivalis strain ATCC33277 OMV using a strategy including ultracentrifugation, sonication, and successive anionic and cationic fast protein liquid chromatography (FPLC). The presence and purity of the protease were confirmed by SDS-PAGE and detection of protease activity using fluorogenic substrates. Rat antibodies produced against the unique adhesin hemagglutinin (H1) domain of RgpA (amino acids 719–865) were titrated by ELISA at a 1:100 dilution using whole P. gingivalis lysate as an antigen and western blotting to detect a 75 kDa band corresponding to RgpA. ResultsDouble anionic-cationic FLPC yielded prominent peaks with evident amidolytic gingipain activity of the appropriate molecular weight, as confirmed by western blotting. The final RgpA yield from 1 L of bacterial culture with colony forming unit (CFU) (Log10) 7.4 ± 0.08/mL was of 12.6% (2 mg/mL), with 3.2 FU/μg of amidolytic activity. ConclusionsThis protocol allows purification of native RgpA from OMV that retains protease activity.