# US 5,700,648 A · Streptolysin O antigen derivatives, their production and uses > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,700,648 A - Document ID: US5700648A - Status: Granted - Kind: A - Application number: 08/296,879 - Filed: 1994-08-26 - Granted: 1997-12-23 - Earliest claimed: 1990-06-25 - Inventors: Michael Kehoe; Michael Pinkney - Web page: https://patentgrep.com/patent/US5700648A ## CPC - C07K 14/315: from Streptococcus (G), e.g. Enterococci - C07K 16/1275: Streptococcus (G) - Y10S 530/806: Antigenic peptides or proteins - Y10S 530/825: Bacteria - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract This application teaches a cytolytic derivative of the thiol-activated protein streptolysin O. The derivatization comprises the deletion of the amino acid cysteine from the amino acid sequence of naturally occurring streptolysin O; end optionally other alterations in said naturally occurring amino acid sequence by amino acid substitution, deletion, inversion, insertion or addition. ## Claims ### Claim 1 A cytolytic derivative of the protein streptolysin O (SLO) immunologically cross-reactive with naturally occurring streptolysin O wherein Cys-530 of naturally occurring SLO has been substituted by a different amino acid. ### Claim 2 (depends on 1) A cytolytic derivative according to claim 1 which is resistant to inactivation by oxidation or by chemical modification of a thiol group. ### Claim 3 (depends on 1) A cytolytic derivative according to claim 1 wherein the different amino acid is alanine. ### Claim 4 (depends on 1) A cytolytic derivative according to claim 1 which comprises part of the amino acid sequence shown in FIG. 2B-1, 2B-2 and 2B-3 and wherein said boxed alanine residue may be substituted by any amino acid other than cysteine. ### Claim 5 A process which comprises the production of a cytolytic derivative of the protein streptolysin O (SLO) immunologically cross-reactive with naturally occurring SLO wherein Cys-530 of naturally occurring SLO has been substituted by a different amino acid by expression of the cytolytic derivative in a recombinant host cell from DNA encoding the cytolytic derivative. ### Claim 6 A method of purifying anti-streptolysin O antibodies which comprises binding them to a cytolytic derivative of the protein streptolysin O (SLO) immunologically cross-reactive with naturally occurring SLO wherein Cys-530 of naturally occurring SLO has been substituted by a different amino acid. ### Claim 7 A method of raising anti-streptolysin O antibodies which comprises immunizing a subject with a cytolytic derivative of the protein streptolysin O (SLO) immunologically cross-reactive with naturally occurring SLO wherein Cys-530 of naturally occurring SLO has been substituted by a different amino acid. ### Claim 8 A diagnostic kit for detecting the presence or absence of antibodies to streptolysin O (SLO) in a clinical sample, which comprises a cytolytic derivative of SLO immunologically cross-reactive with naturally occurring SLO wherein Cys-530 of naturally occurring SLO has been substituted by a different amino acid together with ancillary components for detecting the binding of the derivative to anti-SLO antibodies in the sample. ### Claim 9 A method for detecting the presence or absence of antibodies to streptolysin O (SLO) in a clinical sample, which method comprises contacting the sample with a cytolytic derivative of SLO immunologically cross-reactive with naturally occurring SLO wherein Cys-530 of naturally occurring SLO has been substituted by a different amino acid, and measuring the cytolytic activity remaining after contact between the sample and said cytolytic derivative. ### Claim 10 (depends on 1) A cytolytic derivative according to claim 1 that has the sequence depicted in FIGS. 2B-1, 2B-2 and 2B-3. ### Claim 11 (depends on 1) A cytolytic derivative according to claim 1 that has a serine at position 530. ### Claim 12 (depends on 1) A cytolytic derivative according to claim 1, wherein said streptolysin O (SLO) has the sequence depicted in FIGS. 2A-1, 2A-2 and 2A-3. ### Claim 13 A streptolysin O (SLO) protein derivative lacking the cysteine residue at position 530 of wild-type SLO, but retaining SLO cytolytic activity, wherein said activity is inhibited by neutralizing anti-SLO antiserum. ### Claim 14 A streptolysin O (SLO) derivative lacking the cysteine residue at position 530 of wild-type SLO, but retaining SLO cytolytic activity, wherein said activity is inhibited by cholesterol. ### Claim 15 A cytolytic derivative of streptolysin O (SLO), wherein the SLO has the sequence depicted in FIGS. 2A-1, 2A-2 and 2A-3, wherein at least the cysteine residue at position 530 has been substituted by a different amino acid. ### Claim 16 A method for making a cytolytic derivative of streptolysine O (SLO) comprising substituting at least the codon for the cysteine at position 530 of the naturally occurring SLO of FIGS. 2A-1, 2A-2 and 2A-3 with a codon encoding a different amino acid.