# US 5,707,620 A · Lysine-specific Porphyromonas gingivalis proteinase > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,707,620 A - Document ID: US5707620A - Status: Granted - Kind: A - Application number: 08/541,902 - Filed: 1995-10-10 - Granted: 1998-01-13 - Earliest claimed: 1993-10-21 - Assignees: University of Georgia Research Foundation, Inc. - Inventors: James Travis; Jan Stanislaw Potempa; Robert Neil Pike - Web page: https://patentgrep.com/patent/US5707620A ## CPC - C12N 9/52: derived from bacteria {or Archaea} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Provide 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 exemplified, 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. ## Claims ### Claim 1 A proteinase preparation comprising a substantially pure Lys-gingipain protein complex, said Lys-gingipain having an apparent molecular mass of 105 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis, wherein sample is prepared without boiling, said Lys-gingipain having amidolytic and proteolytic activity for cleavage after L-lysine residues and having no amidolytic and/or proteolytic activity for cleavage after L-arginine residues, wherein the amidolytic and/or proteolytic activity is inhibited by TLCK, glycyl-glycine, 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 trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, EDTA, leupeptin, antipain, serine protease group-specific inhibitors including diisopropylfluorophosphate and phenylmethyl sulfonylfluoride. ### Claim 2 (depends on 1) The proteinase preparation of claim 1 wherein said Lys-gingipain protein complex comprises a catalytic component characterized by an N-terminal amino acid sequence as in SEQ ID NO:1. ### Claim 3 (depends on 1) The proteinase preparation of claim 1 wherein said Lys-gingipain complex has an enzymatically active component characterized by the amino acid sequence as given in SEQ ID NO:14 from amino acid 1 through amino acid 509. ### Claim 4 (depends on 1) The proteinase preparation of claim 1 wherein said preparation comprises proteins of molecular weights of 60, 30, 27 and 17 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after boiling of a sample of said proteinase preparation. ### Claim 5 (depends on 4) The proteinase preparation of claim 4 wherein said 30 and 27 kDa proteins are each identified by an N-terminal amino acid sequence as given in SEQ ID NO:2. ### Claim 6 (depends on 4) The proteinase preparation of claim 4 wherein said 17 kDa protein is characterized by an N-terminal amino acid sequence as given in SEQ ID NO:3. ### Claim 7 (depends on 1) The proteinase preparation of claim 1 wherein said preparation comprises proteins of molecular weights of 60, 44, 27 and 17 kDa as estimated by SDS-PAGE. ### Claim 8 (depends on 7) The proteinase preparation of claim 7 wherein said 60 kDa protein is characterized by an N-terminal amino acid sequence as given in SEQ ID NO:1. ### Claim 9 (depends on 7) The proteinase preparation of claim 7 wherein said 44 and 27 kDa proteins are each identified by an N-terminal amino acid sequence as given in SEQ ID NO:2. ### Claim 10 (depends on 7) The proteinase preparation of claim 7 wherein said 17 kDa protein is characterized by an N-terminal amino acid sequence as given in SEQ ID NO:3. ### Claim 11 (depends on 1) A vaccine comprising a substantially purified Lys-gingipain protein complex preparation of claim 1 and a suitable carrier therefor. ### Claim 12 (depends on 11) The vaccine according to claim 11, further comprising high molecular weight Arg-gingipain. ### Claim 13 (depends on 11) The vaccine according to claim 11 wherein said Lys-gingipain complex catalytic component has an amino acid sequence as given in SEQ ID NO:14 from amino acid 1 through amino acid 509. ### Claim 14 A method of monitoring exposure of an animal to a Lys-gingipain, comprising the steps of: (a) obtaining a sample from the animal; incubating the sample with Lys-gingipain protein complex; or portions thereof under conditions suitable for antibody-antigen interaction; and (b) detecting the presence of antibody-antigen complexes; wherein the presence of antigen-antibody complexes is indicative of exposure of the animal to Lys-gingipain. ### Claim 15 A method of identifying agents that modulate the effect of a Lys-gingipain protein complex on animals comprising the steps of (a) incubating a Lys-gingipain complex with the agent; (b) exposing animal cells sensitive to Lys-gingipain incubated with the agent; (c) determining the ability of Lys-gingipain protein complex incubated with the agent to affect the cells; (d) comparing the effect seen in step c) with the effect of a control sample of a Lys-gingipain complex that has not been incubated with the agent on animal cells susceptible to the Lys-gingipain complex; wherein the agents that modulate the effect of the Lys-gingipain change the activity of the Lys-gingipain complex as compared to the control sample. ### Claim 16 A method of identifying agents that modulate Lys-gingipain protein complex activity comprising the steps of: (a) incubating a substantially pure preparation of a Lys-gingipain complex with the agent; (b) determining the activity of said complex incubated with the agent; and (c) comparing the activity obtained in step b) with the activity of a control sample of said complex that has not been incubated with the agent. ### Claim 17 A method of ameliorating the affects of Lys-gingipain on an animal affected by Lys-gingipain, comprising administering to the animal an effective amount of a physiologically acceptable Lys-gingipain inhibitor. ### Claim 18 An antibody specific for Lys-gingipain or a catalytic component of a Lys-gingipain protein complex wherein said antibody reacts specifically with a protein identified by an amino acid sequence as given in SEQ. ID NO:1.