| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | July 11, 2000 |
| PATENT TITLE |
Fibronectin binding protein |
| PATENT ABSTRACT |
The present invention relates to new recombinant DNA-molecules comprising nucleotide sequences of S. dygalactiae encoding for at least one protein or polypeptide having fibronectin binding property. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | August 1, 1997 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Abrahmsen et al.--Nucl. Acid Res. 14(18):7487-7500 (1986). Chhatwal et al.--Comp. Immunol. Microbiol. Infect. Dis. 10(2):99-108 Abstract (1987). Duggleby et al.--Nuc. Acid. Res. 11(10):3065-3076 (1983). Espersen et al.--Infect. and Immun. 37(2):526-531 (Aug. 1982). Flock et al.--EMBO J. 6(8):2351-2357 (1987). Froman et al.--J. Biol. Chem. 262(14):6564-6571 (1987). Keil-Dlouha et al. --Biochem. Biophys. Acta. 727:115-21 (1983). Lofdahl et al.--Proc. Natl. Acad. Sci. 80:697-701 (Feb. 1983). Mamo et al.--Micro. Pathog. 2(6):417-424 Abstract (1987). McGavin et al.--J. Biol. Chem. 266(13):8343-7 (1991). Myhre et al.--J. Med. Microbiol. 18(2):189-196 Abstract (1984). Myhre--Infect. Immun. 40(1):29-34 (1983). Nuesch et al.--Gene 32:243-249 (1984). Overbeeke et al.--J. Mol. Biol. 163:513-532 (1983). Raja et al.--Infect. Immun. 58(8):2593-8 (1990). Ryden et al.--J. Biol. Chem. 258(5):3396-3401 (Mar. 1983). Sambrook et al.--Molecular Cloning: a Laboratory Manuel, (2d), 6.39-6.43, B.9 (1989). Signas et al.--Proc. Natl. Acad. Sci. 86:699-703 (1989). Switalski et al.--Eur. J. Clin. Microbiol. 1:381-387 (1982). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A pharmaceutical composition for the treatment of infections caused by S. dysgalactiae which comprises at least one protein or polypeptide derived from S. dysgalactiae having fibronectin binding properties together with a pharmaceutically acceptable carrier or diluent. 2. A method for the treatment of infections caused by S. dysgalactiae in mammals, comprising administering to said mammal a therapeutically active amount of at least one fibronectin binding protein or polypeptide derived from S. dysgalactiae, together with a pharmaceutically acceptable carrier or diluent. 3. A method for preventing or treating mastitis in a ruminant, comprising vaccinating a ruminant with a protein derived from S. dysgalactiae in an amount which is effective to elicit production of antibodies against said S. dysgalactiae bacterial strain, wherein said protein comprises an amino acid sequence selected from the group consisting of: Glu Asp Thr Gln Thr Ser Gln Glu Asp Ile Val Leu Gly Gly Pro (SEQ ID NO: 5) Gln Val Ile Asp Phe Thr Glu Asp Ser Gln Pro Gly Met Ser Gly Asn Ser His Thr Ile Thr Glu Asp Ser Lys Pro Ser Gln Glu Asp Glu Val Ile Ile Gly Gly Gln Gly Gln Val Ile Asp Phe Thr Glu Asp Thr Gln Ser Gly Met Ser Gly Asp Asn Ser His Thr Asp Gly Thr Val Leu Glu Glu Asp Ser Lys Pro Ser Gln Glu Asp Glu Val Ile Ile Gly Gly Gln Gly Gln Val Ile Asp Phe Thr Glu Asp Thr Gln Thr Gly Met Ser Gly; and Glu Glu Thr Leu Pro Thr Glu Gln Gly Gln Ser Gly Ser Thr Thr (SEQ ID NO: 6) Val Glu Asp Thr Lys Gly Pro Glu Val Ile Ile Gly Gly Gln Gly Glu Ile Val Asp Ile Glu Glu Asn Leu Pro Thr Glu Gln Gly Gln Ser Gly Ser Thr Thr Glu Val Glu Asp Thr Lys Gly Pro Glu Val Ile Ile Gly Gly Gln Gly Glu Val Val Asp Ile Glu Glu Ser Leu Pro Thr Glu Gln Gly Gln Ser Gly Gly Ser Thr Thr Glu Val Glu Asp. 4. A method for preventing or treating mastitis in a ruminant, comprising vaccinating a ruminant with a protein derived from S. dysgalactiae in an amount which is effective to elicit production of antibodies against said S. dysgalactiae bacterial strain, wherein said protein is encoded by a DNA sequence selected from the group consisting of: CTA GAT ACC TCA GAA AAC AAA AAA TCT GTA ACT GAA AAA GTA ATA ACT (SEQ ID NO: 9) AGC GAT GTT AAA TAT AAG ATT AAT GAT AAA GAA GTG AAA GGT AAA GAA CTA GAC GAT GTC TCT TTA ACT TAC AGT AAA GAA ACC GTT CGT AAG CCA CAG GTG GAA CCA AAT GTT CCT GAT ACA CCT CAG GAA AAA CCA TTG ACA CCG CTT GCA CCG TCA GAA CCT TCA CAA CCA TCT ATT CCA GAG ACA CCA CTG ATA CCG TCA GAA CCT TCA GTT CCA GAG ACA TCA ACA CCA GAA GGT CCA ACA GAG GGA GAA AAT AAT CTT GGT GGT CAG AGT GAA GAG ATA ACG ATT ACA GAA GAT TCT CAA TCA GGG ATG TCT GGT CAA AAT CCT GGT TCT GGA AAT GAA ACA GTG GTT GAA GAC ACT CAA ACA AGT CAA GAG GAT ATT GTA CTT GGT GGT CCA GGT CAA GTG ATT GAC TTT ACA GAA GAT AGC CAA CCG GGT ATG TCT GGT AAT AAT AGC CAT ACT ATT ACA GAA GAT TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT CAA GGT CAG GTG ATT GAC TTT ACA GAA GAT ACT CAA TCT GGT ATG TCT GGG GAT AAT AGC CAT ACA GAT GGG ACA GTG CTT GAA GAA GAC TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT CAA GGT CAA CTG ATT GAC TTT ACA GAA GAT ACC CAA ACC GGT ATG TCT GGG GCT GGA CAA GTA GAG AGT CCA ACA ACT ACC GAA GAA ACC CAT AAA CCA GAA ATA ATC ATG GGC GGT CAA AGT GAC CCT ATT GAT ATG GTT GAG GAC ACT CTT CCT GGT ATG TCT GGC TCT AAT GAA GCT ACT GTT GTG GAA GAA GAC ACA CGT CCT AAA CTT CAA TTC CAT TTT GAT AAT GAA GAG CCC GTT CCT GCA ACG GTT CCA ACC GTT TCT CAA ACT CCT ATT GCT CAG GTA GAA AGT AAA GTG CCT CAT GCC AAA GCA GAG AGT GCG TTA CCT CAA ACT GGA GAT ACA AAT AAA CTA GAA ACG TTC TTT ACC ATT ACA GCA CTA ACT GTT ATT GGA GCG GCA GGA TTA CTA GGC AAA AAA CGT CGT AAT AAT CAA ACT GAT TTA TCA GCA GAT TTC ATC AAA CGC TAT AAA CAA GGC TAA CAT TTT AGC CTT GTT TTA TAT TGT TTC ACT GAC CTC TAA AAG TTA TGA CTG TTT TAA AGG GGG GGT AGG CCA ATC CTC AAA AGT AGT TAA GTT GAG AAA CAC CAC ATC ACT TTA GTC TTA CTG CGC ATA CTA AAA GCA AAA GAT AAT TAG GAG CAG TTG CTA ACT GGA AAA AAT CAA ATG CAA AGC TAG TTG CCA AAG AAC TCT AGA; GAA GAC ACT CAA ACA AGT CAA GAG GAT ATT GTA CTT GGT GGT CCA (SEQ ID NO: 3) CAA GTG ATT GAC TTT ACA GAA GAT AGC CAA CCG GGT ATG TCT GGT AAT AAT AGC CAT ACT ATT ACA GAA GAT TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT CAA GGT CAG GTG ATT GAC TTT ACA GAA GAT ACT CAA TCT GGT ATG TCT GGG GAT AAT AGC CAT ACA GAT GGG ACA GTG CTT GAA GAA GAC TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT CAA GGT CAA CTG ATT GAC TTT ACA GAA GAT ACC CAA ACC GGT ATG TCT GGG; CTC GAG GAA ACT TTG CCA AAC GAG GAA CAT CAA TCA GGT GAT ACC ACT ATT GAA GAT ACT CGC CCG ATT GAT ACC ATG TCA GGT CTA TCA GGA GAG ACT GGG CAG TCT GGT AAT ACT ACA ATT GAG GAA GAT AGT ACG ACT CAC GTT AAA TTC TCA AAA CGT GAT ATT AAT GGT AAA GAA CTA GCA GGT GCT ATG ATT GAA CTA CGT AAT CTA TCA GGT CAA ACT ATT CAA TCA TGG ATA TCA GAC GGC ACA GTT AAA GTT TTC TAC TTG ATG CCA GGG ACT TAT CAA TTT GTG GAG ACG GCA GCG CCA GAA GGT TAT GAA TTG GCA GCT CCA ATT ACC TTC ACA ATT GAT GAG AAA GGA CAA ATT TGG GTA GAC AGT ACA ATT ACT GAG GCG AGT CAA TCT ATT GAT TTC GAG GAA ACT TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACG GAG GTT GAG GAT ACT AAA GGC CCA GAA GTC ATT ATC GGC GGT CAG GGA GAG ATT GTT GAT ATC GAG GAG AAC TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACT GAA GTA GAG GAT ACT AAA GGC CCA GAA GTC ATT ATC GGC GGT CAA GGA GAG GTT GTT GAT ATT GAG GAG AGC TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACT GAA GTA GAA GAT AGC AAG CCT AAA CTC TCT ATC CAC TTT GAT AAC GAG TGG CCT AAG GAA GAC AAA CCA CAA CTA CCT GCC GTT GAA AAA CCT AAG ACT AAG GAG AGC TTG CCA GCC GCA GGG GAA GCT GAA CAT GTC TTA TCT ACT ATC GTG GGA GCA ATG ATC; and GAG GAA ACT TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACG (SEQ ID NO: 4) GTT GAG GAT ACT AAA GGC CCA GAA GTC ATT ATC GGC GGT CAG GGA GAG ATT GTT GAT ATC GAG GAG AAC TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACT GAA GTA GAG GAT ACT AAA GGC CCA GAA GTC ATT ATC GGC GGT CAA GGA GAG GTT GTT GAT ATT GAG GAG AGC TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACT GAA GTA GAA GAT. 5. A composition of matter comprising at least one protein or polypeptide isolated from S. dysgalactiae and a carrier therefor, wherein said protein or polypeptide has fibronectin binding properties. 6. A method for the treatment of infections caused by S. dysgalactiae in mammals, comprising administering to said mammal a therapeutically active amount of at least one fibronectin binding protein or polypeptide derived from S. dysgalactiae |
| PATENT DESCRIPTION |
DESCRIPTION 1. Technical Field The present invention relates to fibronectin binding proteins and hybrid-DNA molecules, e.g., plasmids or phages containing at least one nucleotide sequence encoding for said proteins. Further the invention relates to micro-organisms containing such molecules and their use to produce said proteins, and the synthetic production of said proteins. The object of the present invention is to obtain minimal fibronectin binding proteins. A further object of the present invention is to obtain said proteins by means of genetic engineering technique using e.g., a plasmid containing a nucleotide sequence encoding for the proteins. A further object of the present invention is to obtain a possibility to prepare said proteins by means of chemical synthesis. 2. Background of the Invention WO-A1-85/05553 discloses bacterial cell surface proteins having fibronectin, fibrinogen, collagen, and/or laminin binding ability. Thereby it is shown that different bacteria have an ability to bind to fibronectin, fibrinogen, collagen and/or laminin. It is further shown that fibronectin binding protein from Staphylococcus aureus has a molecular weight of 165 kD, and/or 87 kD, whereby it is probable that the smaller protein is a part of the larger one. Fibronectin is a large glycoprotein having a molecular weight of about 450 kD and having two similar subunits, which can have varying molecular sizes depending on a complex splicing pattern of the precursor mRNA. The protein is present in basement membranes, and connective tissue, but also in a soluble form in different body fluids, such as blood plasma (1). After the original discovery by Kuusela in 1978 that S. aureus binds to fibronectin (2) it has been shown that certain strains of other pathogenic bacteria, such as streptococci of different serological types (3), E. coli (4) and Salmonella (5) can bind to this protein (6). Adhesion of pathogenic bacteria to surfaces is today a generally recognized concept in the discussions of wound pathogens using surface receptors to bind to different proteins on epithelium cell surfaces, in connective tissue matrix, and in wound crusts, such as e.g., fibronectin, fibrinogen, collagen and laminin. The problem is that these receptors are present in a relatively small amount on the bacterial cell surface, and that they are difficult to release. One feasible way in cases the receptors consist of proteins is to clone the genes for the receptors in question to be able to prepare them in quantities which makes it considerably easier to study infections and the course of infections as well as prophylactical and therapeutical treatment of infections by wound pathogens. Screening studies of different serological groups of streptococci, such as A, C, and G according to Lancefield (3) have shown that the strains tested can bind to different connective tissue proteins such as fibronectin, fibrinogen, collagen and laminin and different immunoglobulins (7,8) to a varying degree and with different specificity. In order to further characterize fibronectin binding proteins from streptococci, particularly genes from Streptococcus dysgalactiae for such proteins have been cloned in E. coli. The fibronectin binding domains of these proteins have also been localized and properties and functions of proteins containing these domains will be discussed below. DESCRIPTION OF THE PRESENT INVENTION It has now surprisingly been shown possible to obtain hybrid-DNA molecules comprising nucleotide sequences of the genes coding for proteins or polypeptides having fibronectin binding properties. As evident from the below the following nucleotide sequences [SEQ ID NOS.:1 & 2] are present in the plasmides, pSDF102, and pSDF203, respectively, which encode said proteins. CTA GAT ACC TCA GAA AAC AAA AAA TCT GTA ACT GAA AAA GTA ATA ACT AGC GAT GTT AAA TAT AAG ATT AAT GAT AAA GAA GTG AAA GGT AAA GAA CTA GAC GAT GTC TCT TTA ACT TAC AGT AAA GAA ACC GTT CGT AAG CCA CAG GTG GAA CCA AAT GTT CCT GAT ACA CCT CAG GAA AAA CCA TTG ACA CCG CTT GCA CCG TCA GAA CCT TCA CAA CCA TCT ATT CCA GAG ACA CCA CTG ATA CCG TCA GAA CCT TCA GTT CCA GAG ACA TCA ACA CCA GAA GGT CCA ACA GAG GGA GAA AAT AAT CTT GGT GGT CAG AGT GAA ATA ACG ATT ACA GAA GAT TCT CAA TCA GGG ATG TCT GGT CAA AAT CCT GGT TCT GGA AAT GAA ACA GTG GTT GAA GAC ACT CAA ACA AGT CAA GAG GAT ATT GTA CTT GGT GGT CCA GGT CAA GTG ATT GAC TTT ACA GAA GAT AGC CAA CCG GGT ATG TCT GGT AAT AAT AGC CAT ACT ATT ACA GAA GAT TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT CAA GGT CAG GTG ATT GAC TTT ACA GAA GAT ACT CAA TCT GGT ATG TCT GGG GAT AAT AGC CAT ACA GAT GGG ACA GTG CTT GAA GAA GAC TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT CAA GGT CAA GTG ATT GAC TTT ACA GAA GAT ACC CAA ACC GGT ATG TCT GGG GCT GGA CAA GTA GAG AGT CCA ACA ACT ACC GAA GAA ACC CAT AAA CCA GAA ATA ATC ATG GGC GGT CAA AGT GAC CCT ATT GAT ATG GTT GAG GAC ACT CTT CCT GGT ATG TCT GGC TCT AAT GAA GCT ACT GTT GTG GAA GAA GAC ACA CGT CCT AAA CTT CAA TTC CAT TTT GAT AAT GAA GAG CCC GTT CCT GCA ACG GTT CCA ACC GTT TCT CAA ACT CCT ATT GCT CAG GTA GAA AGT AAA GTG CCT CAT GCC AAA GCA GAG AGT GCG TTA CCT CAA ACT GGA GAT ACA AAT AAA CTA GAA ACG TTC TTT ACC ATT ACA GCA CTA ACT GTT ATT GGA GCG GCA GGA TTA CTA GGC AAA AAA CGT CGT AAT AAT CAA ACT GAT TAA TCA GCA GAT TTC ATC AAA CGC TAT AAA CAA GGC TAA CAT TTT AGC CTT GTT TTA TAT TGT TTC ACT GAC CTC TAA AAG TTA TGA CTG TTT TAA AGG GGG GGT AGG CCA ATC CTC AAA AGT AGT TAA GTT GAG AAA CAC CAC ATC ACT TTA GTC TTA CTG CGC ATA CTA AAA GCA AAA GAT AAT TAG GAG CAG TTG CTA ACT GGA AAA AAT CAA ATG CAA AGC TAG TTG CCA AAG AAC TCT AGA and/or CTC GAG GAA ACT TTG CCA AAC GAG GAA CAT CAA TCA GGT GAT ACC ACA ACT ATT GAA GAT ACT CGC CCG ATT GAT ACC ATG TCA GGT CTA TCA GGA GAG ACT GGG CAG TCT GGT AAT ACT ACA ATT GAG GAA GAT AGT ACG ACT CAC GTT AAA TTC TCA AAA CGT GAT ATT AAT GGT AAA GAA CTA GCA GGT GCT ATG ATT GAA CTA CGT AAT CTA TCA GGT CAA ACT ATT CAA TCA TGG ATA TCA GAC GGC ACA GTT AAA GTT TTC TAC TTG ATG CCA GGG ACT TAT CAA TTT GTG GAG ACG GCA GCG CCA GAA GGT TAT GAA TTG GCA GCT CCA ATT ACC TTC ACA ATT GAT GAG AAA GGA CAA ATT TGG GTA GAC AGT ACA ATT ACT GAG GCG AGT CAA TCT ATT GAT TTC GAG GAA ACT TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACG GAG GTT GAG GAT ACT AAA GGC CCA GAA GTC ATT ATC GGC GGT CAG GGA GAG ATT GTT GAT ATC GAG GAG AAC TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACT GAA GTA GAG GAT ACT AAA GGC CCA GAA GTC ATT ATC GGC GGT CAA GGA GAG GTT GTT GAT ATT GAG GAG AGC TTA CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACT GAA GTA GAA GAT AGC AAG CCT AAA CTC TCT ATC CAC TTT GAT AAC GAG TGG CCT AAG GAA GAC AAA CCA CAA CTA CCT GCC GTT GAA AAA CCT AAG ACT AAG GAG AGC TTG CCA GCC GCA GGG GAA GCT GAA CAT GTC TTA TCT ACT ATC GTG GGA GCA ATG ATC whereby the smaller repetitive regions (cf. FIG. 3) in each gene above code for the peptides having fibronectin binding activity. The invention further comprises a plasmid or phage comprising a nucleotide sequence coding for said fibronectin binding proteins. The invention further comprises micro-organisms containing at least one hybrid-DNA molecule according to above. Such micro-organisms have been deposited at the Deutsche Sammlung von Mikroorganismen under deposition number DSM 4614 (pSDF102) and DSM 4613 (pSDF203). The invention further relates to a process for preparing fibronectin binding proteins comprising transfer of at least one hybrid-DNA molecule according to above into a micro-organism, cultivating the said micro-organism in a culture medium, and isolating the protein thus formed in a manner known per se. A further aspect of the present invention comprises a chemical synthesis of the fibronectin binding proteins, whereby amino acids connected into peptides in which the order of amino acids is based upon said nucleotide sequences encoding said proteins. The synthesis starts from the C-terminal glycine, and aspartic acid, respectively, which are reacted stepwise with the appropriate amino acid, whereby they are finally reacted with glutamic acid, and glutamic acid, respectively, at the N-terminal end to the formation of the fibronectin binding peptide regions. Appropriate amino acids can also be fused to said amino acid sequence such as the IgG binding region of protein A. The invention will be described more in detail in the following with reference to the Examples given, however, without being restricted thereto. |
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| PATENT PHOTOCOPY | Available on request |
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