Purification and Characterization of Gingipains

Gingipains are cysteine proteases produced in large quantities by Porphyromonas gingivalis which together constitute important virulence factors in the pathogenesis of periodontal disease by that organism. Described is this unit is an efficient procedure for the purification of gingipains from the growth medium of P. gingivalis strain HG66, along with detailed protocols for growth of the organism and basic characterization of the purified proteases. The purification procedure consists of acetone precipitation followed by gel filtration to separate high-molecular-mass gingipains (Kgp and HRgpA) from RgpB. Kgp and HRgpA are further separated on Arg-Sepharose by the virtue of differential elution from the affinity matrix with lysine (Kgp) and arginine (HRgpA) eluant. Obtained from these procedures, the gingipains are stable and can be stored at -80 degrees C for years without loss of activity.