IE83742B1 - Fibronectin binding protein - Google Patents

Description

PATENTS ACT, 1992 1558/89 FIBRONECTIN BINDING PROTEIN ALFA-LAVAL AGRI INTERNATIONAL AKTIEBOLAG DESCRIPTION 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. is to obtain minimal The object of the present invention 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 possi- bility to prepare said proteins by means of chemical synthesis.

Background of the invention W0-A1-85/05553 discloses bacterial cell surface proteins having fibronectin, fibrinogen, collagen, and/or Laminin binding abi- lity. Thereby lity to bind to fibronectin, fibrinogen, collagen it is shown that different bacteria have an abi- and/or lami- nin. 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 kb 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 mem- in a soluble form in (1). branes, and connective tissue, but also different body fluids, After the ori- such as blood plasma ginal discovery by Kuusela in 1978 that g; aureus binds to fib- ronectin (2) it has been shown that certain strains of other pathogenic bacteria, such as streptococci of different serolo- gical types (3), E; coli (4) and Salmonella (S) can bind to this protein (6).

Adhesion of pathogenic bacteria to surfaces is today a general- Ly recognized concept in the discussions of wound pathogens using surface receptors to bind to different proteins on epi- thelium 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 therapeuti- cal treatment of infections by wound pathogens.

Screening studies of different serological groups of strepto- cocci, 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 potypeptides having fibronectin binding properties.

SEQUGFTCES are DFESEFVC As evident from the below the foLLoHing nucteotide respectivety, which encode said proteins.

CTA AGC CTA CAG CCG CTG CCA ACA AAT GAA CAA AAT GAA GGT *GAG GAT GAT GAC GTG CTT ATA ACA GAA GAA GAC GTG AGC GAT CAG AAT GAC CAA ACC GTT GAT GAA GCA CCG GAG GAT ACA ACT ATT CAT TCT GTG AGC TCT GTG TCA AAA GTC CCA CCG TCA GGA TCT GTG CAA GAC ACT AAA ATT CAT AAA ATT GAA TAT TCT AAT TCA GAA GAA CAA GTT ACA TTT ATT CCA GAC ACA CCA GAC AAC AAG TTA GTT GAA CCT AAT TCA AGT ACA ACA AGT TTT GAT AGT TTT AAA ATT ACT CCT CCT TCA AAT GGG CAA GAA CAA ACA GGG CAA ACA AAA AAT TAC GAT TCA GTT CTT ATG GAG GAT GAA ACA GAG GAA TCT GAT AGT ACA CAA CCA GGT TCT GAT AGC GAT GAT GTG GAT GAT GTA AAA AAA CCT CCA GAG GGT GGT ATT CAA GAG ACT CTT GAG ACC ACT GAA GAA CAG TCT ACA CAG CAA GTA CCG GTG CAA GAA GTG CAA GAA GTG ACC GAA ATT TCA AGT AAT CTT GGT ATA TCT ATA ACC AAA AAA GTT AAA CCA ACA GTA GGT CGT CCA GAG CCA GAA*ATA GGT ATG ATC GGT ATC GGT GGT TCT GGC ATG GGC ATG ATA AAA AAG TTG ACA GAA ACG TCT CCA GGT GGT TCT GGT TCT in the ptasmides, pSDF1D2, and pSDF203, ACT GAA CCA ACA CCA GGT ATT GGA GGT AAT CAA GGG CAA GGG GCT GAA ACT GAC CCT AGT GAT ATT GAT AGC TAA CAC GAG AAC GGA ATA CTT ACA GCA AAA ACA GGA TAA CTT AGG ATC CAG TCT and/or CTC ACT GAG CAC GCT ATA CAA ATT ATT GAG GTT ATT GAG GTA GTT GAG GTA GAG ATT ACT GTT ATG TCA TTT ACC ACT GAA GAG GTT GAG GAG GTT GAG GAA CAA ATC CCT CGT ACG GTG AAT GCG TCA GTT GGG ACT TTG AGA GAA GAA GGG AAA ATT GAC GTG TTC GAG ACT GAT GAT AAC GAT GAT AGC GAT GTA ATG GGT CCT GTT CCT AAA GCA GCA TTA GGT TTA CTA ACT GAT CAG TTC GAA GGC GAG ACA GCG TTA ACT ATC GAG GGC ATG AAA CCA CAT CTA GGA GAT TAT AGG GTC ACT TTG ACT TCT TCA CTA ACA ACG ATT AGT CCA AAA CCA AAA AGT GGT TCT CTT ACC GCC GAA TTC TGT CCA TTA GGA CCA CGC GGT AAA CGT GTT GCA GAT CAA ACT GGC ACT GGC CCA CAA GGC CAA GTT AAA ACG CTA ATC TTC ATC CTG AAA AAC cce AAT CGT AAT AAA GCG GAG TCT GAA CCA GAA CCA ACA AGT TCT TTC TCT GCA TTC GGC AAA ACT CTC CGC AAT GAG ATT ACT GAT CTA GTT CCA AAA ATT CAA GAA CAA GAA ACT GAC AAT CAT CAA GAG TTT AAA CGC GAC AAA ATA CAA GAA GAT ACA ATT TCA TTC GAA GGA GAT GGC GTC GGC GTC ACC CCT GAA TTT ACT AGT ACC AAA TAT CTC AGT CTA ATG CAT ACC ATT AAT GGT TAC GGT CAA TTC CAA ATT CAA ATT GAA ATT GCT GAT CCT GCG ATT CGT AAA TAA AGT AAA CAA CAA ATG AGAG GGT CAA TTG TAT ATT TCT ATC TCT ATC GAA GAT ACT AAT ATT TTA ACA CGT CAA AAG TAA GCA AGC TCA TCA GAA AAA ACT ATG GAA TGG GGC GGC GGC GGC ACC ATG GTT GAA GCT CCT GCA AAT GGC TTA GTT AAA TAG GGT GGT GAT GAA ATT CCA TTG GTA TCT GGT TCT GGT CAT GTT GTG GAG CAG CAA CTA AAT TAA TGA GAG GAT TTG GAT CTA AGT CTA CAA GGG GCA GAC ACA CAG ACA CAA AAA GAG GAA CCC GTA ACT ACT CAA CAT CTG AAA AAT CCA ACC TCA ACG GCA TCA ACT GCT AGT ACG GGA ACT GGA CCA GAC GAA GTT GAA GGA GTT ACT TTT TTT CAC TAG AAG ACA GGA ACT GGT TGG TAT CCA ACA GAG GAG GAA GAG CCT AAA CTC CCA CAA CTA GCC GCA GGG ATC AGC GAC TTG GCA AAG AAA CCA ATG TCT ATC CAC TTT GAT AAC GAG TGG CCT GCC GTT GAA AAA CCT AAG ACT GAA GCT GAA CAT GTC TTA TCT ACT CCT AAG GAA AAG GAG AGC ATC GTG GGA whereby the smaLLer (cf. 3) the peptides having fibronectin binding repetitive regions Fig. in each gene above code for activity.

The invention further comprises a ptasmid or phage comprising a nucLeotide sequence coding for said fibronectin binding pro- teins.

The invention further comprises micro-organisms containing at Least one hydrid-DNA moLecuLe according to above. Such micro- organisms have been deposited at the Deutsche Sammtung von Mik- roorganismen under deposition number DSM 4614 (pSDF102) and DSM 4613 (pSDF203).

The invention further retates to a process for preparing fibro- nectin 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 isotating the protein thus formed in a manner known per se.

A further aspect of the present invention comprises a chemicat 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-terminat glycine, and aspartic acid, respectiveLy, which are reacted stepwise with the appro- priate amino acid, whereby they are finaLLy reacted with gLuta- mic 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.

Example 1 Construction of a gene bank of chromosomal DNA from Streptococ- cus dysgalactiae Chromosomal DNA from Streptococcus dysgalactiae, strain 52, was prepared in accordance with the perchlorate method (9). The DNA was partially cleaved using £33 3A1, was size fractionated on a 1% agarose gel, and the DNA fragment within the size range 3 to 9 kb were collected, electro eluated, and purified on a Nensorb (Du Pont) column.

The plasmid vector pUC18 was cleaved using E33 HI and was phos- phatase treated. The partially cleaved and fractionated strep- tococcus-DNA was ligated with the cleaved pUC18 vector. The li- gation mixture was transformed to freeze competent E; Egli, strain T61, and was spred on LA plates containing ampicillin (50 /ug/ml) and IPTG (0.1mM), and 0.004% X-gal, called axi- plates. white colonies were transferred to LA plates with am- picillin (50 /ug/ml).

Screening of a gene bank for a fibronectin binding protein (FNBP) The white colonies from the axi plates were picked using tooth picks to LA plates with ampicillin, 52 colonies per plate. In total 728 transformants were collected. These were screened with regard to fibronectin binding activity using a filter assay method according to below.

Transformants are picked from an axi-plates to LA plates with ampicillin, and the plates are incubated over night. From these plates the colonies are replicated over to new LA plates, and which are incubated at 37°C over night. A nitro-cellulose fil- -6... ter is put onto each agarplate with grown out colonies. when the filters are completely moistened the colonies are attached by suction and the filters are carefully removed. The filters are exposed to chloroform vapour for 5 min, and are then wash- ed, 3 x 10 min, 37°c in a buffer solution consisting of 100mM Tris-HCL pH 7.5, 0.05 X Tween-40, and 1S0mM NaCl. The filters The 10mM Tris-HCL pH 7.5, and 1.4% fat free milk powder, for 2 hrs at 37°C, or room tem- are allowed to dry at room temperature for about 30 min. filters are preincubated in 1SOmM Nacl, perature over night. The milk powder buffer has to be freshly 125 is added (about 30,000 cpm prepared. I labelled fibronectin incubated at room temperature per filter), and the filters are over night. The filters are washed, 3 x 10 min at 370C using a and 150mM NaCl, An unexposed film The film solution of 0.05 X Tween-40, whereupon the fil- ters are dried. is put thereon, and is expos- ed for 3 to 5 days. 125 is developed and the clones which have bound to I-fibronectin are identified and isolated.

The filter screening assay showed 3 positive clones, which all were further analysed. The fibronectin binding ability was fur- ther determined in a competition assay (10). Lysate of the E; coli clones were prepared by Lysing the bacteria using lyso- zyme (1 mg/ml) in a buffer solution consisting of 100 mM Tris- HCl pH 7.5, 1SOmM NaCl, and 1mM EDTA. acti-vity was analysed by determining the ability of the lysa- The fibronectin binding with regard to their ability to bind to the inhibited by adding a lysate of E; coli clone containing a gene for fibronectin binding protein of E; EUFEUS.

Restriction mapping and subcloning Plasmid-DNA of the three positive subclones from the filter assay, called pSDF100, pSDF200, the LiCl method (11) 6.5 kb, and analysis on agarose gels. and pSDF3OO were prepared using and determined to be 4.9 kb, 6.9 kb, and respectively, by cleavages using restriction enzymes All three clones were cleaved using about 20 of the most common restriction enzymes, which recognizes a sequence of 6 nucleotides and starting from cleavage pattern restriction maps were drafted. Two of the clones, pSDF100, and pSDF3OD, were partly overlapping having a .9 kb sequence in common, and thus only one was selected for further studies. As pSDF10D had a higher fibronectin binding activity than pSDF30O the former was selected. p$DF100 and pSDF200 in order to were subcloned identify more closely the regions encoding fibronectin binding activity. pSDF1OO was cleaved using Bam HI, whereupon the plasmid was re- fied and The other two Xbal-XbaI fragments were puri- inserted into the pUC18 vector. One of these fragments encodes fibronectin binding activity. This clone was called pSDF1D2. from pSDF200. The Elal-EEEI fragment deleted from pSDF200 gave a clone called pSDF201, and further the gglll-EEERI fragment eliminated from pSDF201 gives pSDF202. Finally, the XhoI-EcoRI In the corresponding way subclones were constructed fragment has been deleted from pSDF202. This new clone was the- reby obtained was called pSDF203. All these new subclones are positive, i.e., they express fibronectin binding activity, cf.

FIG 1a and FIG. 1b.

Further subcloning by EcoIII digestion In order to facilitate the nucleotide sequencing according to the dideoxymethod smaller subclones differing 150 to 200 base pairs in length are required in order to obtain overlapping DNA sequence. Exonucleas III digest one of the DNA strands from the ‘ overhang, or from the blunt end, but leaves the 3' overhang.

The single stranded DNA is then digested using S1-nuclease. in the is used "Erase-a-Base" USA) This technique System Kit (Promega, Madison, and makes it possible to construct series of subclones which differs in some hundreds of interest the fibronectin Table 1 below.

In cases of cf. nucleotides in size. binding activity was tested, Table 1 Inhibition assay in tubes Assay mixture: 100 /ul of lysate of E; coli clones containing streptococcal DNA clones (the bacteria were grown on LB + 50 ug ampicillin + 1mM IPTG, / washed, and concentrated to 0D54D = 5.0) 100 /ul Cowan I cells, heat killed, OD " 5.0 125 540 100 /ul I Labelled fibronectin, 8865 cpm 200 /ul PBS + 0.1 X BSA Incubation: 2 hrs, room temperature washing: Twice in P85 + 0.1 X BSA + 0.05 Z Tween The resutts are evident from Table 1 below.

Lysate of Ditution of Number of cpm Z binding in relation subclone Lysate to controL without Lysate ControL without Lysate 4430 100 pSDF102c10 undil 550 12.4 '2 3870 87.4 pSDF102c13 undiL 200 4.5 '2 1440 32.5 pSDF102c9 undiL 610 13.8 '2 3170 71.6 pSDF102c11 undil 1400 31.6 '? 3490 78.8 pSDF102c14 undit 630 14.2 '2 3220 72.7 pSDF102c18 undiL 4030 91.0 '2 4300 97.1 pSDF20303 undiL 640 14.4 '2 2780 62.8 pSDF203c6 undiL 2710 61.2 ’2 4790 108 pSDF203c8 undiL 3180 71.8 ’2 3660 82.6 p$DF203c11 undil 3540 79.9 '2 3970 89.6 pSDF203c1S undit 3860 87.1 '? 4300 97.1 pSDF203c9 undii 4020 90.7 '2 4730 107 pSDF102 undiL 200 4.5 '2 1050 23.7 pSDF203 undiL 180 4.1 ’2 950 21.4 TG1 undit 3690 83.3 Nucleotide sequencing Subctones obtained after an exolll digestion and other subcLo- nes were sequenced using the dideoxy method according to Gem SeqR dsDNA Sequ-encing System (Promega Biotech., Madison, USA) Nucleotide sequencing of pSDF102 gave the fotlowing CTA GAT ACC TCA GAA AGC CTA CAG CCG CTG CCA ACA AAT GAA CAA AAT GAA GGT GAT GAA GGT GAT GAC GTG CTT ATA ACA GAA GAA GAC GTG AGC GAT CAG AAT GAC CAA * GAG GTT GAT GAA GCA CCG GAG GAT ACA ACT ATT CAT TCT GTG AGC TCT GTG AAA GTC CCA CCG TCA GGA TCT GTG CAA GAC ACT AAA ATT CAT AAA ATT TAT TCT AAT TCA GAA GAA CAA GTT ACA TTT ATT CCA GAC ACA CCA GAC AAC AAA AAA TCT AAG TTA GTT GAA CCT AAT TCA AGT ACA ACA AGT TTT GAT AGT TTT ATT ACT CCT CCT TCA AAT GGG CAA GAA CAA ACA GGG CAA ACA AAT TAC GAT TCA GTT CTT ATG GAG GAT GAG GAA ACA GAG GAA GAT AGT ACA CAA CCA GGT TCT GAT AGC GAT GAT GTG GAT GAT GTA AAA AAA CCT CCA GAG GGT GGT ATT CAA GAG ACT CTT GAG ACC ACT GAA AAA GAA GAA CAG TCT ACA CAG CAA GTA CCG GTG CAA GAA GTG CAA GTG ACC GAA ATT TCA AGT AAT CTT GGT ATA TCT ATA ACC AAA GTT AAA CCA ACA eAA*ATA GGT ATG ATC GGT ATC GGT sequence: GTA ATA ACT" GGT CGT CCA GAG CCA AAA AAG TTG ACA GAA ACG TCT GAA CCA ACA CCA GGT ATT GGT GGA GGT TCT CCA GGT GGT AAT GGC ATG GGT TCT CAA GGG GGC ATG GGT TCT CAA GGG GCT GAA ACT GAC CCT AGT GAT ATT GAT AGC TAA CAC GAG AAC GGA ATA CTT ACA GCA AAA ACA GGA TAA CTT AGG ATC CAG TCT whereby GAA CAA AAT GAC GTG AGC CAA ATC CCT CGT ACG GTG AAT GCG TCA GTT GGG ACT TTG AGA GGT CAA GTG GTA ATG GGT CCT GTT CCT AAA GCA GCA TTA GGT TTA CTA GAG GGC ATG AAA CCA CAT CTA GGA GAT TAT AGG GTC ACT AGT GGT TCT CTT ACC GCC GAA TTA TTC TGT CCA TTA GGA CCA ACA AGT TCT TTC TCT GCA TTC GGC AAA ACT CTC CGC AAT ACT GAC AAT CAT CAA GAG TTT AAA CGC GAC AAA ATA CAA CAA GGC CAA GTT AAA ACG CTA ATC TTC ATC CTG AAA ACC CCT GAA TTT ACT- AGT ACC AAA TAT CTC AGT CTA ATG GAA ATT GCT GAT CCT GCG ATT CGT AAA TAA AGT AAA CAA GAA GAT ACT AAT ATT TTA ACA CGT CAA AAG TAA GCA AGC repetitive domains of the sequence CAA GAC ACT ACA TTT ATT AGT ACA ACA ATT GAC TTT encode a peptide having GAA GAT AGC CAA ACA GGG GAG GAA ACA GAT GAT GTG CAA GAG GAT ACA GAA GAT fibronectin GAG ACT CTT GAG ACC GAG GAT ATT GTA GTG CAA GAA GTG CAA CTT GGT ATA TCT ATA ACC ACC ATG GTT GAA GCT CCT GCA AAT GGC TTA GTT AAA TAG CAT GTT GTG GAG CAG CAA CTA AAT TAA TGA GAG GAT TTG GGT ATG GGT TCT ATC GGT GGC ATG ATC GGC GGT ATG binding activity.

AAA GAG GAA CCC GTA ACT ACT CAA CAT CTG AAA AAT CCA GGT TCT GGT TCT CCA GAC GAA GTT GAA GGA GTT ACT TTT TTT CAC TAG AAG CAA GGG CAA GGG CTC ACT GAG CAC GCT ATA CAA ATT ATT GAG GTT ATT GAG GTA GTT GAG GTA AGC GAC TTG GCA nucteotide sequencing of pSDF203 gave the foLLowing GAG ATT ACT GTT ATG TCA TTT ACC ACT GAA GAG GTT GAG GAG GTT GAG GAA AAG AAA CCA ATG GAA GAA GGG AAA ATT GAC GTG TTC GAG ACT GAT GAT AAC GAT GAT AGC GAT CCT CCA GCC ATC ACT GAT CAG TTC GAA GGC GAG ACA GCG TTA ACT ATC TTA ACT ATT CAA GCA TTG ACT TCT TCA CTA ACA ACG ATT AGT CCA AAA CTC CTA GGG CCA CGC GGT AAA CGT GTT GCA GAT CAA ACT GGC TCT CCT GAA AAC CCG AAT CGT AAT AAA GCG GAG TCT GAA CCA ATC GCC GCT GAG ATT ACT GAT CTA GTT CCA AAA ATT CAA GAA CAC GTT GAA GAA GAT ACA ATT TCA TTC GAA GGA GAT GGC GTC TTT GAA CAT CAT ACC ATT AAT GGT TAC GGT CAA TTC CAA ATT GAT AAA GTC CAA ATG GAG GGT CAA TTG TAT ATT TCT ATC TCT ATC AAC CCT TTA TCA TCA GAA AAA ACT ATG GAA TGG GGC GGC eec sec‘ GAG AAG TCT GGT GGT GAT GAA ATT CCA TTG GTA TCT GGT TCT GGT TGG ACT ACT GAT CTA AGT CTA CAA GGG GCA GAC ACA CAG ACA CAA CCT AAG ATC SGQUEFI“ ACC TCA ACG GCA TCA ACT GCT AGT ACG GGA ACT GGA AAG GAG GTG ACA GGA ACT GGT TGG TAT CCA ACA GAG GAG GAA GAG GAA AGC GGA whereby the repetitive domains of the sequence TTA ACT GAG GTT ATT GAA GAG GTT ACT GAT GAT CCA ACT GAA CAA AAA GGC CCA GAA GGC CAA GTC ATT TCT GGC ATC GGC TCT GGT ACA CAG ACG GGA GAG GAG TTA ACT ATT GAG GTA GTT GAG GAG GTT AAC GAT GAT CCA AAA ACT GGC GAA CCA CAA GAA GGC GTC CAA ATT TCT ATC GGC GGC TCT GGT ACA CAA ACT GGA GAA GAG GAG TTA GAG GAA AGC GAT CCA ACT GAA CAA GGC CAA TCT GGC TCT ACA ACT GAA encode a peptide having fibronectin binding activity.

Southern bLot hybridisation detects no homoLogies on DNA LeveL between the genes for the fibronectin binding protein of E; au- The inhibition between the proteins from the respective binding. western btot anatyses of Lysate of the two fibronectin binding Labelled fibronectin E; coLi clones studied indicate using and autoradiography shows that subctone pSDF203 encodes a pro- tein having a motecular weight of 70 kDa, and subclone pSDF102 a corresponding protein having a motecular weight of 110 kD.

The deduced amino acid sequences des from the above given nucleot the following: Gln Thr Phe Thr Glu Asp Ser Thr Glu Gln Asp Thr Gln Val Ile Thr Ser His Ile Gln Thr Glu Glu Thr Ser Phe Asp Ser Pro Glu Gly Asp Lys Ile Asp Gln Asp Asn Ser His Thr Gln Thr Glu Glu Ser Phe Lys Pro Glu Gly Gln Asp Thr Gly Glu Gln Thr Leu Pro Thr Glu Glu Val Glu Val Ile Asp Lys Pro Thr Gly Glu Gln Leu Thr Glu Glu Val Glu Asn Val Asp Lys pro Ser Leu Pro Thr Glu Gln Glu Glu Glu Val Asp, respectively.

Asp Asp Val Asp Asp Gly Val Gly Val the proteins or polypepti- sequences encode for are Ile Gln Glu Thr Glu Thr Gln Ile Gln Ile Val Gln Glu Val Gln Ser Ile Ile Thr Gly Gly Gly Gly Gly Met Ile Gly Ile Gly Ser Gly Gly Met Gly Met Thr Gln Thr Gln The present fibronectin binding proteins can be used in Asn* Gln Gly Gln Gly Thr Gly Glu Glu Thr Gly Glu Glu Thr Thr immuni- zation, whereby the proteins, preferably in combination with a fusion protein in order to form a larger antigen to react upon, are injected in doses creating an immunological reaction in the host mammal. Thus the fibronectin binding proteins can be used in vaccination of rumens to mastitis created by streptococcal infections.

*Asn Further, the fibronectin binding proteins can be used to block an infection in an open skin Lesion. wounds can be treated by using a suspension comprising the fibronectin binding protein.

Thus the fibronectin binding proteins can be used to treat in fibro- wounds, for blocking bacterial binding sites e.g., nectin, or for immunization (vaccination). In the latter case the host produces specific antibodies which can protect against attachment by bacterial strains comprising such fibronectin binding proteins. Hereby the antibodies block the adherence of the bacterial strains to damaged tissue.

Examples of colonizing of tissue damage are: a) colonizing of wounds in skin and connective tissue, which wounds have been caused by a mechanical trauma, chemical da- mage, and/or thermical damage; mouth cavity, colonizing of wounds on mucous membranes such as in the or in the mammary glands, urethra or vagina; c) colonizing of connective tissue proteins, which have been in connection exposed by minimal tissue damage (micro lesions) with epithelium and endothelium (mastitis, heart valve infec- tion, hip exchange surgery). when using the present fibronectin binding proteins, prepared by means of hybrid-DNA technique, or synthesized, for immuniza- tion (vaccination) in mammals, including humans, the proteins, or polypeptides are dispersed in sterile isotonic saline solut- ion, optionally while adding a pharmaceutically acceptable dis- persing agent. Different types of adjuvants can further be used in order to sustain the release in the tissue, and thus expose the protein for a longer period of time to the immuno defence system of a body. is 0.5 to S ronectin binding protein per kg body weight and A suitable dose to obtain immunization /ug of fib- injection at In order to obtain durable vaccina- immunization. immunization, tions should be carried out at consecutive occasions with an interval of 1 to 3 weeks, preferably at three occasions. Adju- vants are normally not added when repeating the immunization treatment. when using the present fibronectin binding proteins or polypep- tides for local topical administration the protein is dispersed in an isotonic saline solution to a concentration of 25 to 250 ug per ml. The wounds are then treated with such an amount on- ly to obtain a complete wetting of the wound surface. For an average wound thus only a couple of millilitres of solution are used in this way. After treatment using the protein solution the wounds are suitably washed with isotonic saline solution or another suitable wound treatment solution. fibronectin binding protein of the present invention lized on a solid carrier, such as small latex or SepharoseR beads, whereupon sera containing antibodies are allowed to pass and react with the fibronectin binding protein thus immobiliz- The agglutination is then measured by known methods.

Further the fibronectin binding protein or polypeptide can be used in an ELISA test (Enzyme Linked Immuno Sorbent Assay; E Engvall, Med. Biol. 55, 193 (1977)). Hereby wells in a poly- styrene microtitre plate are coated with the fibronectin bind- ing protein and incubated over night at 4°C. The plates are then thoroughly washed using PBS containing 0.05% Tween 20, and dried. Serial dilutions of the patient serum made in PBS-Tween, are added to the wells, and are incubated at 30°C for 1.5 hrs.

After rinsing anti-human IgG conjugated with an enzyme, or a horseradish peroxidase, or an alkaline phosphatase is added to the wells and further incubated at 30°C for 1.5 hrs. During these incubations IgG from patient serum, and added antihuman IgG-enzyme conjugate, respectively, has been bound thereto.

After rinsing, is added, an enzyme substrate p-nitrophosphate -‘]7_ in case of an alkaline phosphatase, or orthophenylene diamine substrate COPD) in case a peroxidase has been used, respective- ly. The wells of the plates are then rinsed using a citrate buffer containing 0.055% OPD, and 0.005% H202, °C for 10 min. The enzyme reaction is stopped by adding a AN and incubated at solution of H2504 to each well. The colour development is mea- sured using a spectrophotometer.

Depending on the type of enzyme substrate used a fluorescence measurement can be used as well.

Another method to diagnoze E; dysgalactiae infections is by us- ing the DNA gene probe method based on the nucleotide sequence Thereby is attached to a solid for the fibronectin binding protein or part thereof. the natural or synthetic DNA sequence carrier, such as a nitrocellulose filter, a nylon filter, or a in The DNA gene probe, optionally labelled enzymatically, or by a radioactive polystyrene plate as mentioned above, by e.g., adding a milk the case of diagnozing a mastitis, to the surface. isotope, is then added to the solid surface plate comprising the DNA sequence, whereby the DNA gene probe attaches to the membrane associated sequence where appearing. The enzyme or radioactive isotope can readily be determined by known methods.

Above the term fibronectin binding protein includes any of the polypeptide sequences as well, which constitute the minimal fibronectin binding site of the complete protein.

LEGENDS TO THE FIGURES FIG.

FIG.

Restriction map A. Restriction map of the clone.

. Different subcLones constructed to determine the region in the gene which codes for fibronectin binding activity. The binding activity of the different gene products have been indicated.

C. Subclones obtained after digestion with ExoIII of pSDF102, and pSDF203, 100 H is the part of the DNA sequence which encodes the (=COOH- respectively. Scale: 1 cm = bp. membrane associated part of the protein Subclone p102c1O a). A1, A2 och A3, respectively, denote repetitive domains of the FIG. 3) contains the 3' end of the and B1, B2, and B terminal). gene (FIG. 3, sequences (cf.

Inhibition assay in tubes at the addition of lysates of E; coli-clones. The percentage values given are related to the binding of in the absence of I labelled fibronectin to cells Lysate. As a negative control a lysate of E; coli TG1 which had no influence on the binding of the cells to fibronectin. with pUC18—vector without insert was used, E; coli clone 015 contains a gene from E; aureus encoding for fibronectin binding activity. shows repetitive sequences of pSDF102 och pSDF203. shows the nucleotide and deducted amino acid sequences of pSDF102 shows the nucleotide and deducted amino acid sequences of pSDF2 . . 6.

References 1. 2. 3.

Hymes, R.0. (1985) Annu. Rev. CeLL Biol. 1, 67-90.

Kuuseta, P. (1978) Nature gig, 718-720.

SwitaLski, L. et at (1982) Eur. J. CLin. Microbiol. 1, 381-387.

Froman, G. et at. (1984) J. Biol. Chem. 252, 14899-14905.

Batoda, S.B. et at (1985) FEMS Microbiot. Lett. 28, 1-5. wadstrom, T. et aL (1985) in Jackson, G.J. (ed), Pathogenesis of Infection, Springer Verlag, Bertin, Heidetberg, New York, Tokyo, pp. 193-207. b Lopes, J.D. et at (1985) Science 229, 275-277.

Langone, 1.1. (1982) Adv. Immunot. 32, 157-252.

Marmur, J. (1961) J. Mot. Biol. 3, 208-218.

FLock, J.-I. et aL (1987) The EMBO Journat Q, Monstein, H.-J. et aL (1986) Biochem. Int. 12, 889-896. -2357.

Claims (12)

1. Claims: Plasmid or phage comprising a nucleotide sequence sequence reading either 1) GAA GGT GAA CAA TCT GAA CAA TCT GAC CAA AAT GAT GGT GGG GAC GGT GGG and/or 2) GAG GAG GGA GAG GAA GAG GAA GAA GTT GAG GAG GTA GAG GAG GTA ACT CAA ACA AGT CAA GAG GAT ATT GTG ATT GAC TTT ACA GAA GAT AGC AAT AGC CAT ACT ATT TCT AAA CCA AGT CAA CAG GTG ATT GAC TTT GAT AAT AGC CAT ACA TCT AAA CCA AGT CAA CAA GTG ATT GAC TTT ACT GAG ATT GAG GTT AGC GAA ACA GAT GAG GAA GAT GGG ACA GAG ACA GAT GAG ACA GAT GAG GAA GAT TTA CCA ACT GAA CAA GGC CAA GAT ACT AAA GGC CCA GAA GTC GTT GAT ATC GGC CAA GAA GTC CCA ACT GAA CAA ACT AAA GGC CCA GAT ATT TTA GAT GTT GGC CAA TTA CCA ACT GAA CAA GAT. dysgalactiae encoding a protein or a polypeptide said nucleotide GTA CAA CTT CCG ATA CAA CTT GTG ACT GTG GTG ACC ATA CAA TCT GGC ATT ATC TCT ATT ATC TCT GGT CCA ATG TCT GGT GGT GGC GGT GGT ATG ATC TCT GAA ATC ACC GGC GGT GGT ATG TCT ACA ACG GGC GGT CAG TCT ACA ACT GGC GGT CAA TCT ACA ACT
2. 2. the GAA GGT GGT GAA CAA TCT GAA CAA TCT Plasmid or phage according to claim 1,1), wherein nucleotide sequence reads GAC ACT CAA ACA AGT CAA GAG GAT ATT GTA CTT GGT GGT CCA CAA GTG ATT GAC TTT ACA GAA CAT AGC CAA CCG GGT ATG TCT AAT AAT AGC CAT ACT ATT ACA GAT TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT GGT CAG GTG ATT GAC TTT ACA GAA GAT ACT CAA TCT GGT ATG GGG GAT AAT AGC CAT ACA GAT GGG ACA GTG CTT GAA GAC TCT AAA CCA AGT CAA GAG GAT GAG GTG ATA ATC GGC GGT GGT CAA GTG ATT GAC TTT ACA GAA GAT ACC CAA ACC GGT ATG GGG.
3. Plasmid or phage according to claim 1,2), wherein the nucleotide sequence reads 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.
4. 4. Plasmid pSDF102 as contained in the E. coli TGl strain, having the deposit number DSM 4614.
5. 5. Plasmid pSDF203 as contained in the E. coli TGl strain, having the deposit number DSM 4613.
6. 6. E. coli strain expressing at least one of said fibronectin binding proteins according to claims l—3.
7. 7. Micro-organism transformed by the recombinant DNA molecule according to one or more of claims 1-5.
8. 8. Plasmid or phage comprising one or more of the the nucleotide sequences according to claim 1-3.
9. 9. Micro—organism comprising at least one plasmid or phage according to claim 8.
10. l0. Process for the preparation of a fibronectin binding protein or polypeptide, whereby a) at least one plasmid according to claims 1-3 is introduced into a micro—organism, b) said micro—organism is cultured in a growth promoting medium, and c) the protein thus formed is isolated in a manner known per se.
11. ll. A fibronectin binding protein or polypeptide comprising at least one of the amino acid sequences Glu Asp Thr Gln Thr Ser Gln Glu Asp Ile Val Leu Gly Gly Pro Gly 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. *Asn 20
12. 12. A fibronectin binding protein or polypeptide comprising at least one of the amino acid sequences Glu Glu Thr 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 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. l3. Pharmaceutical composition for the treatment of infections caused by S. dysgalactiae, whereby it comprises at least one fibronectin binding protein as defined by the nucleotide sequences of claims l—3, and/or amino acid sequences of claims ll—12 together with a pharmaceutically acceptable carrier or diluent. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS