| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | December 22, 1998 |
| PATENT TITLE |
Collagen binding protein as well as its preparation |
| PATENT ABSTRACT |
The present invention relates to a new recombinant DNA-molecule comprising a nucleotide sequence from S. aureus coding for a protein, or polypeptide, having collagen binding properties. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | May 22, 1995 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Patti et al., J. Biol. Chem. 267: 4766-4772 (1992). Patti et al., J. Biol. Chem. 269: 11672 (1994). Switalski et al., J. Biol. Chem. 264: 21080-21086 (1989). Hunkapiller et al., Meth. Enzymol. 91: 227-236 (1983). Lathe, J. Mol. Biol. 183: 1-12 (1985). Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985). Genbank Accession Number M81736. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A plasmid pSAC104 as contained in the E. coli TG1 having the deposit number DSM 6199. 2. An E. coli strain expressing the protein encoded by the plasmid of claim 1. 3. A microorganism transformed by said plasmid of claim 1. 4. An isolated DNA-molecule characterized in that it comprises the following nucleotide sequence ›SEQ ID NO: 1!: |
| PATENT DESCRIPTION |
DESCRIPTION 1. Technical Field The present invention relates to a collagen binding protein as well as hybrid-DNA-molecules, e.g. plasmids or phages comprising a nucleotide sequence coding for said protein. Further the invention relates to microorganisms comprising said molecules and their use producing said protein, as well as the synthetic preparation of said protein. In particular the invention relates to a cloned gene encoding the Staphylococcus aureus collagen binding protein, or functionally active portions thereof, vectors containing the cloned gene or parts thereof, and microorganisms transformed by those vectors as well as the cloning of the gene which specify the biosynthesis of Staphylococcus aureus collagen binding protein (CBP) (also called the collagen receptor by Switalski et al 1989) and the use of organisms transformed with the cloned gene to produce CBP or CBP like proteins. The invention also describes the use of this gene for diagnostic purposes. The object of the present invention is to obtain a collagen binding protein. A further object is to obtain said protein by means of a genetic engineering technique by using e.g. a plasmid comprising a nucleotide sequence coding for said protein. A further object is to obtain a possibility of preparing said protein by chemical synthesis. Further objects will be apparent from the following description. 2. Background of the Invention WO-Al-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. Regarding the binding of collagen to S. aureus several studies have been reported (Carret et al 1985, Holderbaum et al 1985, Holderbaum et al 1986, Vercellotti et al 1985, Speziale et al 1986, Switalski et al 1989). Switalski et al 1989 reported on the isolation and characterization of a S. aureus surface protein which they identified as a collagen receptor. Using lysostaphin to release the protein from the cell wall followed by ion exchange chromatography, ammonium sulfate precipitation and gel filtration it was possible to purify a protein with an apparent Mr of 135 kDa. It was also shown that antibodies raised against the 135 kDa protein inhibited the binding of collagen to S. aureus Cowan 1 cells. DESCRIPTION OF THE INVENTION It has now surprisingly been found possible to obtain a hybride-DNA-molecule comprising a nucleotide sequence coding for a protein or a polypeptide having collagen binding properties. As evident from below the following nucleotide sequence ›SEQ ID NO: 1! is present in the gene coding for said protein. ATGCACTTGT ATTCGTTATA CTGTATATAT TTTGCATAAT AAAATAATAA TATGAATTTT TGATAAATTT CATTGAATAA GAACTAAATT AGTTTATAAT TTATTATTAG TATCCTGTGG ATATGACATA GAGTATAAGG AGGGGTTTTT ATGAACAAAA ATGTGTTGAA GTTTATGGTC TTTATAATGT TATTAAATAT CATCACACCT TTATTTAATA AAAATGAAGC ATTTGCAGCA CGAGATATTT CATCAACGAA TGTTACAGAT TTAACTGTAT CACCGTCTAA GATAGAAGAT GGTGGTAAAA CGACAGTAAA AATGACGTTC GACGATAAAA ATGGAAAAAT ACAAAATGGT GACATGATTA AAGTGGCATG GCCGACAAGC GGTACAGTAA AGATAGAGGG TTATAGTAAA ACAGTACCAT TAACTGTTAA AGGTGAACAG GTGGGTCAAG CAGTTATTAC ACCAGACGGT GCAACAATTA CATTCAATGA TAAAGTAGAA AAATTAAGTG ATGTTTCGGG ATTTGCAGAA TTTGAAGTAC AAGGAAGAAA TTTAACGCAA ACAAATACTT CAGATGACAA AGTAGCTACG ATAACATCTG GGAATAAATC AACGAATGTT ACGGTTCATA AAAGTGAAGC GGGAACAAGT AGTGTTTTCT ATTATAAAAC GGGAGATATG CTACCAGAAG ATACGACACA TGTACGATGG TTTTTAAATA TTAACAATGA AAAAAGTTAT GTATCGAAAG ATATTACTAT AAAGGATCAG ATTCAAGGTG GACAGCAGTT AGATTTAAGC ACATTAAACA TTAATGTGAC AGGTACACAT AGCAATTATT ATAGTGGACA AAGTGCAATT ACTGATTTTG AAAAAGCCTT TCCAGGTTCT AAAATAACTG TTGATAATAC GAAGAACACA ATTGATGTAA CAATTCCACA AGGCTATGGG TCATATAATA GTTTTTCAAT TAACTACAAA ACCAAAATTA CGAATGAACA GCAAAAAGAG TTTGTTAATA ATTCACAAGC TTGGTATCAA GAGCATGGTA AGGAAGAAGT GAACGGGAAA TCATTTAATC ATACTGTGCA CAATATTAAT GCTAATGCCG GTATTGAAGG TACTGTAAAA GGTGAATTAA AAGTTTTAAA ACAGGATAAA GATACCAAGG CTCCTATAGC TAATGTAAAA TTTAAACTTT CTAAAAAAGA TGGATCAGTT GTAAAGGACA ATCAAAAAGA AATTGAGATT ATAACAGATG CAAACGGTAT TGCTAATATT AAAGCGTTGC CTAGTGGAGA CTATATTTTA AAAGAAATAG AGGCGCCACG ACCGTATACA TTTGATAAGG ATAAAGAATA TCCGTTTACT ATGAAAGATA CAGATAATCA GGGATATTTT ACGACTATTG AAAATGCAAA AGCGATAGAA AAAACAAAAG ATGTTTCTGC TCAAAAGGTT TGGGAAGGCA CTCAAAAAGT GAAACCAACG ATTTATTTCA AGTTGTACAA ACAAGATGAC AATCAAAATA CAACACCAGT AGACAAAGCA GAGATTAAAA AATTAGAAGA TGGAACGACA AAAGTGACAT GGTCTAATCT TCCGGAAAAT GACAAAAATG GCAAGGCTAT TAAATATTTA GTTAAAGAAG TAAATGCTCA AGGTGAAGAT ACAACACCAG AAGGATATAC TAAAAAAGAA AATGGTTTAG TGGTTACTAA TACTGAAAAA CCAATCGAAA CAACATCAAT TAGTGGTGAA AAAGTATGGG ACGACAAAGA CAATCAAGAT GGTAAGAGAC CAGAAAAAGT CAGTGTGAAT TTATTGGCTA ACGGGGAGAA AGTAAAAACG TTAGACGTGA CATCTGAAAC AAACTGGAAG TACGAATTTA AAGACTTACC GAAGTATGAT GAAGGAAAGA AAATAGAATA TACAGTGACC GAAGATCACG TAAAAGACTA CACAACAGAC ATCAACGGTA CGACAATAAC GAACAAGTAT ACACCAGGAG AGACATCGGC AACAGTAACA AAAAATTGGG ATGACAATAA TAACCAAGAC GGAAAACGAC CAACTGAAAT CAAAGTTGAG TTATATCAAG ATGGAAAAGC AACAGGAAAA ACGGCAATAT TAAATGAATC TAATAACTGG ACACATACGT GGACAGGATT AGATGAAAAA GCAAAAGGAC AACAAGTAAA ATACACAGTC GAGGAATTAA CAAAGGTCAA AGGTTATACA ACACATGTGG ATAACAATGA TATGGGTAAC TTGATTGTGA CGAATAAATA TACGCCAGAA ACAACATCAA TTAGTGGTGA AAAAGTATGG GACGACAAAG ACAATCAAGA TGGTAAGAGA CCAGAAAAAG TCAGTGTGAA TTTATTGGCT GATGGAGAGA AAGTAAAAAC GTTAGACGTG ACATCTGAAA CAAACTGGAA GTACGAATTT AAAGACTTAC CGAAGTATGA TGAAGGAAAG AAAATAGAAT ATACAGTGAC CGAAGATCAC GTAAAAGACT ACACAACAGA CATCAACGGT ACGACAATAA CGAACAAGTA TACACCAGGA GAGACATCGG CAACAGTAAC AAAAAATTGG GATGACAATA ATAACCAAGA CGGAAAACGA CCAACTGAAA TCAAAGTTGA GTTATATCAA GATGGAAAAG CAACAGGAAA AACGGCAATA TTAAATGAAT CTAATAACTG GACACATACG TGGACAGGAT TAGATGAAAA AGCAAAAGGA CAACAAGTAA AATACACAGT CGAGGAATTA ACAAAGGTCA AAGGTTATAC AACACATGTG GATAACAATG ATATGGGCAA CTTGATTGTG ACGAATAAAT ATACGCCAGA AACAACATCA ATTAGTGGTG AAAAAGTATG GGACGACAAA GACAATCAAG ATGGTAAGAG ACCAGAAAAA GTCAGTGTGA ATTTATTGGC TAACGGAGAG AAAGTAAAAA CGTTAGACGT GACATCTGAA ACAAACTGGA AGTACGAATT TAAAGACTTA CCGAAGTATG ATGAAGGAAA GAAAATAGAA TATACAGTGA CCGAAGATCA CGTAAAAGAC TACACAACAG ACATCAACGG TACGACAATA ACGAACAAGT ATACACCAGG AGAGACATCG GCAACAGTAA CAAAAAATTG GGATGACAAT AATAACCAAG ACGGAAAACG ACCAACTGAA ATCAAAGTTG AGTTATATCA AGATGGAAAA GCAACAGGAA AAACGGCAAT ATTAAATGAA TCTAATAACT GGACACATAC GTGGACAGGA TTAGATGAAA AAGCAAAAGG ACAACAAGTA AAATACACAG TCGATGAATT AACAAAAGTT AATGGCTATA CAACGCATGT GGATAACAAT GATATGGGTA ACTTGATTGT GACAAATAAA TATACGCCGA AAAAACCGAA TAAACCAATC TATCCTGAAA AACCAAAAGA CAAAACACCA CCAACTAAAC CTGATCATTC TAATAAAGTT AAACCAACTC CCCCAGATAA GCCATCAAAA GTGGATAAGG ATGATCAACC TAAAGATAAT AAAACCAAAC CTGAAAATCC TCTAAAAGAA TTACCAAAAA CTGGTATGAA GATTATAACT TCATGGATTA CATGGGTATT TATAGGTATA TTGGGACTGT ATTTAATTTT AAGAAAAAGA TTTAACTCAT AAACCATTAT AATTATTTTT ATAGATAAGG CTATTCTTAG TTCTATGTAT AATACATGTA TATTAATAGG TCACTTTTAA TCTGTATGTA AGCAGACTAA GAGTGGCCTT TTAAACAAAT AAAAAAA whereby this nucleotide sequence encodes for the following protein starting at nucleotide no.151 in the reading above, whereby the prepresent nucleotides ›SEQ ID NO: 2! shown in FIG. 2 are part of the signal system: Ala ArgAspIleSerSerThrAsnValThrAspLeuThrValSerProSerLysIleGluAsp GlyGlyLysThrThrValLysMetThrPheAspAspLysAsnGlyLysIleGlnAsnGly AspMetIleLysValAlaTrpProThrSerGlyThrValLysIleGluGlyTyrSerLys ThrValProLeuThrValLysGlyGluGlnValGlyGlnAlaValIleThrProAspGly AlaThrIleThrPheAsnAspLysValGluLysLeuSerAspValSerGlyPheAlaGlu PheGluValGlnGlyArgAsnLeuThrGlnThrAsnThrLeuAspAspLysvalAlaThr IleThrSerGlyAsnLysSerThrAsnValIleGlyTrpIleLysvalLysArgGluPro ValValPheLeuIleAsnLysSerGlyLysIleCysTyrGlnGluAspThrThrHisVal ArgTrpPheLeuAsnIleAsnAsnGluLysSerTyrValSerLysAspIleThrIleLys AspGlnIleGlnGlyGlyGlnGlnLeuAspLeuSerThrLeuAsnIleAsnValThrGly ThrHisSerAsnTyrTyrSerGlyGlnSerAlaIleThrAspPheGluLysAlaPhePro GlySerLysIleThrValAspAsnThrLysAsnThrIleAspValThrIleProGlnGly TyrGlySerTyrAsnSerPheSerIleAsnTyrLysThrLysIleThrAsnGluGlnGln LysGluPheValAsnAsnSerGlnAlaTrpTyrGlnGluHisGlyLysGluGluValAsn GlyLysSerPheAsnHisThrValHisAsnIleAsnAlaAsnAlaGlyIleGluGlyThr ValLysGlyGluLeuLysValLeuLysGlnAspLysAspThrLysAlaProIleAlaAsn ValLysPheLysLeuSerLysLysAspGlySerValValLysAspAsnGlnLysGluIle GluIleIleThrAspAlaAsnGlyIleAlaAsnIleLysAlaLeuProSerGlyAspTyr IleLeuLysGluIleGluAlaProArgProTyrThrPheAspLysAspLysGluTyrPro PheThrMetLysAspThrAspAsnGlnGlyTyrPheThrThrIleGluAsnAlaLysAla IleGluLysThrLysAspValSerAlaGlnLysValTrpGluGlyThrGlnLysValLys ProThrIleTyrPheLysLeuTyrLysGlnAspAspAsnGlnAsnThrThrProValAsp LysAlaGluIleLysLysLeuGluAspGlyThrThrLysValThrTrpSerAsnLeuPro GluAsnAspLysAsnGlyLysAlaIleLysTyrLeuValLysGluValAsnAlaGlnGly GluAspThrThrProGluGlyTyrThrLysLysGluAsnGlyLeuValValThrAsnThr GluLysProIleGluThrThrSerIleSerGlyGluLysValTrpAspAspLysAspAsn GlnAspGlyLysArgProGluLysValSerValAsnLeuLeuAlaAsnGlyGluLysval LysThrLeuAspValThrSerGluThrAsnTrpLysTyrGluPheLysAspLeuProLys TyrAspGluGlyLysLysIleGluTyrThrValThrGluAspHisValLysAspTyrThr ThrAspIleAsnGlyThrThrIleThrAsnLysTyrThrProGlyGluThrSerAlaThr ValThrLysAsnTrpAspAspAsnAsnAsnGlnAspGlyLysArgProThrGluIleLys ValGluLeuTyrGlnAspGlyLysAlaThrGlyLysThrAlaThrLeuAsnGluSerAsn AsnTrpThrHisThrTrpThrGlyLeuAspGluLysAlaLysGlyGlnGlnValLysTyr ThrValGluGluLeuThrLysValLysGlyTyrThrThrHisValAspAsnAsnAspMet GlyAsnLeuIleValThrAsnLysTyrThrProGluThrThrSerIleSerGlyGluLys ValTrpAspAspLysAspAsnGlnAspGlyLysArgProGluLysvalSerValAsnLeu LeuAlaAspGlyGluLysValLysThrLeuAspValThrSerGluThrAsnTrpLysTyr GluPheLysAspLeuProLysTyrAspGluGlyLysLysIleGluTyrThrValThrGlu AspHisValLysAspTyrThrThrAspIleAsnGlyThrThrIleThrAsnLysTyrThr ProGlyGluThrSerAlaThrValThrLysAsnTrpAspAspAsnAsnAsnGlnAspGly LysArgProThrGluIleLysValGluLeuTyrGlnAspGlyLysAlaThrGlyLysThr AlaThrLeuAsnGluSerAsnAsnTrpThrHisThrTrpThrGlyLeuAspGluLysAla LysGlyGlnGlnValLysTyrThrValGluGluLeuThrLysValLysGlyTyrThrThr HisValAspAsnAsnAspMetGlyAsnLeuIleValThrAsnLysTyrThrProGluThr ThrSerIleSerGlyGluLysValTrpAspAspLysAspAsnGlnAspGlyLysArgPro GluLysValSerValAsnLeuLeuAlaAsnGlyGluLysValLysThrLeuAspValThr SerGluThrAsnTrpLysTyrGluPheLysAspLeuProLysTyrAspGluGlyLysLys IleGluTyrThrValThrGluAspHisValLysAspTyrThrThrAspIleAsnGlyThr ThrIleThrAsnLysTyrThrProGlyGluThrSerAlaThrValThrLysAsnTrpAsp AspAsnAsnAsnGlnAspGlyLysArgProThrGluIleLysValGluLeuTyrGlnAsp GlyLysAlaThrGlyLysThrAlaIleLeuAsnGluSerAsnAsnTrpThrHisThrTrp ThrGlyLeuAspGluLysAlaLysGlyGlnGlnValLysTyrThrValAspGluLeuThr LysValAsnGlyTyrThrThrHisValAspAsnAsnAspMetGlyAsnLeuIleValThr AsnLysTyrThrProLysLysProAsnLysProIleTyrProGluLysProLysAspLys ThrProProThrLysProAspHisSerAsnLysValLysProThrProProAspLysPro SerLysValAspLysAspAspGlnProLysAspAsnLysThrLysProGluAsnProLeu LysGluLeuProLysThrGlyMetLysIleIleThrSerTrpIleThrTrpValPheIle GlyIleLeuGlyLeuTyrLeuIleLeuArgLysArgPheAsnSer In the single letter amino acid sequence above the following abbreviations have been used A Ala, Alanine R Arg, Arginine N Asn, Asparagine D Asp, Aspartic acid C Cys, Cysteine C Cys, Cystine G Gly, Glycine E Glu, Glutamic acid Q Gln, Glutamine H His, Histidine I Ile, Isoleucine L Leu, Leucine K Lys, Lysine M Met, Methionine F Phe, Phenylalanine P Pro, Proline S Ser, Serine T Thr, Threonine W Trp, Tryptophan Y Tyr, Tyrosine V Val, Valine The invention further comprises a plasmid or phage comprising a nucleotide sequence coding for said collagen binding protein. The invention further comprises a microorganism containing at least one hybrid-DNA-molecule according to the above. The plasmid pSAC104 in an E. coli strain TG1 has been deposited at the Deutsche Sammlung von Mikroorganismen (DSM), and has thereby been allocated the deposition number SM 6199. The present invention provides a cloned gene encoding the CBP having improved CBP-properties as compared with native CBP which is released and purified from S. aureus cells. The gene is derived from a S. aureus strain and inserted into a cloning vector. Cells of a procaryotic organism which have been transformed with recombinant vectors are disclosed. The invention further provides the identification of the nucleotide sequence of the gene encoding the CBP here called the cbp-gene. The deduced amino acid sequence reveals a molecule with several distinct features resembling staphylococcal cell surface proteins. The invention also provides a procedure for production and purification of the recombinant CBP. This is done in a way so that the molecule retains its collagen binding properties, thus this recombinant CBP resemblance the native unreleased S. aureus CBP. The invention further provides the use of the cbp-gene for diagnostic purposes. Gene probes chosen to specifically recognize the presence of the cbp gene in clinical S. aureus isolates have been used. As an example, the correlation between the presence of CBP on the surface of S. aureus strains isolated from patient with septic arthritis could be verified by the presence of the cbp-gene in all tested strains. Appropriate carrier proteins can be coupled to the amino acid sequence as well, such as IgG binding regions of protein A. The invention will be described in the following with reference to the examples given, however, without being restricted thereto. BRIEF DESCRIPTION OF THE FIGURES FIG. 1: (A), Simplified restriction map of the insert in p 16 and cCOLR6A showing the region of homology, MCS is an abbreviation for multi cloning site. (B), Schematic drawing of the cbp-gene encoding the different regions. S is the proposed signal sequence followed by region A and the repetitive B regions, W is the cell wall spanning region and M the membrane anchoring region. FIGS. 2A-2I ›SEQ ID NO: 8!: Nucleotide sequence and the deduced amino acid sequence of the assembled sequence from the insert in p 16 and cCOLR6A. The different regions are marked by arrows and sequences resembling ribosomal binding sites (RBS). The 5'end and 3'end of the insert in p 16 as well as the 5'end of the insert in cCOLR6A is indicated. FIG. 3: Western blot of lysates of clinical isolates of S. aureus probed with anti-collagen adhesin antibodies. Lysostaphin lysates of strains were separated by gel electrophoresis, electroblotted onto an Immobilon-P membrane. Lanes a: Cowan, b: #7, c: #12, d: #13, e: #14, f: #15, g: #16, h: Phillips, and i: #9. FIG. 4: Time dependent attachment of collagen adhesin positive and negative strains of S. aureus to collagen (panel A) and cartilage (panel C). Inhibition of this attachment by anti-adhesin antibodies (panels B and D, collagen and cartilage, respectively). .sup.125 I-labeled cells of two collagen adhesin positive strains--S. aureus Phillips (.DELTA.) and #14 (O) and one adhesin negative strain--#9 (.circle-solid.) were incubated with collagen coated wells or with pieces of cartilage for indicated periods of time. FIG. 5: Binding of .sup.125 I-labeled collagen or adhesion to cartilage by polystyrene beads coated either with the collagen adhesin (O) or a recombinant form of the S. aureus fibronectin receptor (.circle-solid., ZZFR). Panel A--binding of .sup.125 I-collagen to protein coated beads as a function of time. Panel B--inhibition of binding of .sup.125 I-collagen by antibodies. Attachment of .sup.125 I-labeled beads to cartilage as a function of time (panel C) and inhibition of attachment of .sup.125 I-labeled beads to cartilage by antibodies (panel D). In this experiment 1 ug of adhesin protein was coupled to 10.sup.8 polystyrene beads. Control beads were coated with the same molar concentration of the fibronectin receptor. Unreacted sites on the beads were saturated with bovine serum albumin. FIG. 6: Expression constructs utilized to localize the collagen binding domain within the S. aureus collagen adhesin. |
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| PATENT PHOTOCOPY | Available on request |
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