| abstract |
Provided herein is a substantially pure Lys-gingipain complex preparation, Lys-gingipain being characterized as having an apparent molecular mass of 105 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, where sample is prepared without boiling, said Lys-gingipain having amidolytic and proteolytic activity for cleavage after lysine residues and having no amidolytic and/or proteolytic activity for cleavage after arginine residues, wherein the amidolytic and/or proteolytic activity is inhibited by TLCK, cysteine protease group-specific inhibitors including iodoacetamide and iodoacetic acid, wherein the amidolytic and/or proteolytic activity of said Lys-gingipain is not sensitive to inhibition by leupeptin, antipain, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, serine protease group-specific inhibitors including diisopropylfluorophosphate and phenylmethyl sulfonylfluoride, and antibodies specific for the Lys-gingipain protein complex and its catalytic component, methods for preparation. As specifically examplified, a Lys-gingipain protein complex is purified from Porphyromonas gingivalis H66, and the 60 kDa catalytic component of the Lys-gingipain protein complex has an amino acid sequence as given in SEQ ID NO: 14 from amino acid 1 through amino acid 509. Also provided are nucleic acid sequences encoding this catalytic protein. The nucleotide coding sequence of the 60 kDa catalytic component of the Lys-gingipain protein complex is given in SEQ ID NO: 13, from nucleotide 1336 through nucleotide 2863. The Lys-gingipain complex also comprises a hemagglutinin component identified by an N-terminal amino acid sequence as given in SEQ ID NO: 14, amino acids 510-714. |