WO1994010330A9 - Proteine de liaison de la fibronectine et sa preparation - Google Patents

Proteine de liaison de la fibronectine et sa preparation

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Publication number
WO1994010330A9
WO1994010330A9 PCT/US1993/010547 US9310547W WO9410330A9 WO 1994010330 A9 WO1994010330 A9 WO 1994010330A9 US 9310547 W US9310547 W US 9310547W WO 9410330 A9 WO9410330 A9 WO 9410330A9
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WO
WIPO (PCT)
Prior art keywords
protein
fibronectin binding
binding protein
polypeptide
fibronectin
Prior art date
Application number
PCT/US1993/010547
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English (en)
Other versions
WO1994010330A1 (fr
Filing date
Publication date
Application filed filed Critical
Priority to AU55907/94A priority Critical patent/AU692140B2/en
Publication of WO1994010330A1 publication Critical patent/WO1994010330A1/fr
Publication of WO1994010330A9 publication Critical patent/WO1994010330A9/fr
Priority to NO951679A priority patent/NO951679L/no
Priority to FI952108A priority patent/FI952108A/fi

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Definitions

  • the present invention relates to a fibronectin binding protein as well as a hybrid-DNA-molecules, e.g., plasmids and 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.
  • the object of the present invention is to obtain a minimal fibronectin binding protein.
  • a further object of the present invention 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.
  • WO-A1-85/05553 discloses bacterial cell surface proteins having fibronectiri, 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 derived 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.
  • Fibronectins are a family of high molecular weight glycoproteins occurring in a soluble form in many body fluids and in an insoluble form as a compound of cell surfaces, basement membranes, and extracellular matrices. Fibronectins appear to fulfill a critical role in clearance by phagocytes of autologous tissue debris, immune complexes, and bacteria. Fibronectins also
  • SUBSTITUTE SHEET bind to epithelial cells. In doing so it may serve as a receptor for organisms like group A streptococci, but may also shield the epithelial receptors for other organisms. Thus the inability of Gramnegative organisms like Ps. aeuruginosa to colonize the oral cavity of healthy humans may be due to an interference in binding to epithelial receptors by fibronectin. The ability to resist binding to soluble fibronectin has been thought to be a virulence factor in invasive infection by group B streptococci. A number of Grampositive bacterial species including Staphylococcus aureus. other staphylococcus species, and group A, C and G streptococci exhibit specific interaction with fibronectin.
  • F £ ⁇ H can express a variety of adhesins with differing binding specificities. The majority of these adhesins recognize carbohydrate moieties present on glycoconjugates.
  • R. coli may also express binding to matrix proteins such as fibronectin, laminin, and collagen.
  • Uropatogenic R coK expressing the 075 X adhesin bind tubular basement membranes and to the Bowman capsule known to be rich in laminin. The purified 075 X adhesin was specifically found to bind laminin.
  • R coli isolated from patients with ulcerous colitis frequently bind matrix proteins.
  • R coli isolates from bovine mastitis have been shown to bind to fibronectin at a high frequency. Below a native fibronectin binding protein from ⁇ eoli is disclosed, as well as the cloning of the fibronectin binding, fnbA gene from a bovine isolate of R coh that express curli pili and fibronectin binding when present in R coli HB101.
  • Chemically fibronectin is a large glycoprotein (Mj about 450 kD) with two similar subunits, which may vary in molecular size depending on a complex splicing pattern of a precursor MRNA.
  • the major function of fibronectin which is found in body fluids blood clots and extracellular matrices seems to be related to the ability of the protein to mediate substrate adhesion of most eukaryotic cells.
  • AACTCTGCAC TTGCTCTGCA AACTGATGCC CGTAACTCTG ACTTGACTAT TACCCAGCAT GGCGGCGGTA ATGGTGCAGA TGTTGGTCAG GGCTCAGATG
  • the mature curlin protein has a molecular weight of 17 kD when gel- purified, and when determined via the nucleotide sequence it is predicted to contain 122 amino acids providing for a molecular weight of 14,345 dalton.
  • the invention further comprises a plasmid or phage comprising a nucleotide sequence coding for said fibronectin binding protein.
  • the invention further comprises a micro-organism comprising at least one hybrid-DNA-molecule according to the above.
  • the microorganism, R, coli HB101/pFnb20 encompassing the plasmid encoding for said nucleotide sequence was deposited May 05, 1988 at Deutsche Sammlung von
  • the invention further comprises a method for producing a fibronectin binding protein whereby at least one hybrid-DNA-molecule of above is introduced into a micro-organism, cultivating said micro-organism in a growth medium, and isolating the protein thus formed and expressed by means of an affinity chromatography on a fibronectin bound to an insolubilized carrier followed by ion exchange chromatography.
  • a further aspect of the invention comprises a chemical synthesis of the fibronectin binding protein, whereby an amino acid sequence is built up based on said nucleotide sequence encoding for said protein starting from the C- terminal amino acid which is stepwise reacted with the appropriate amino acid, whereby it is finally reacted with the amino acid at the N-terminal end, to form the fibronectin peptide region.
  • carrier proteins can be coupled to the amino acid sequence as well, such as IgG binding regions of protein A.
  • Bacteriophage transducing particles carrying portions of the strain AO12 genome cloned into cosmid vector pJB8 were used to transform R coli HB101.
  • Strain AO12 expressed fibronectin binding when grown on CFA plates at 30°C, but to a lesser extent at 37°C. Transductants were thus
  • the subclone pFnblO was digested with a series of restriction endonucleases to prepare a restriction map as shown in FIG. 1.
  • pFnblO expressing fibronectin binding
  • several subclones were constructed from pFnblO as were various deletion derivatives. These constructs were tested for their ability to confer binding to R. coli HB101.
  • the Hpal and Sphl sites delineates the 1.2 kb region required for fibronectin binding as shown in FIG. 1.
  • the recombinant plasmids shown in FIG. 1 were transformed into the minicell producing strain AA10. Plasmid encoded polypeptides were analyzed from 35 S-methionine labelled minicells.
  • Plasmid pFnblO expressed two polypeptides with molecular masses of 43 kD and 17 kD respectively as shown in FIG 2.
  • pFnb30 is a deletion derivative of pFnblO that lacks a 0.9 kb Clal fragment. This derivative no longer confer fibronectin binding to R coli HB101.
  • the 49 kD polypeptide was still expressed from this clone but the smaller 17 kD polypeptide was missing.
  • the larger polypeptide must be encoded from a gene positioned between the Clal 2 and Kpnl sites on pFnblO, since the region between Sphlj and Clal, site is to small (0.4 kb) to encompass a gene coding for a 49 kD protein.
  • HB101/pFnb20 in an omnimixer and such partially purified preparations contained a dominating protein species with a molecular weight of 17 kD.
  • the 17 kD polypeptide was elektroeluted onto an Immonobulon R filter and the aminoterminal sequence determined by sequential Edman degradation.
  • the isolated protein was the same 17 kD gene product of the structural gene spanning the Clal. and the Hpal sites in pFnb20 this region was sequenced.
  • One open reading frame consisting of 133 codons was identified spanning the Clal and Hpal sites.
  • the protein sequence of the 5 amino terminal residues was identical to the DNA sequence specified by codons 2-6 in the open reading frame confirming that the open reading frame identified encodes the subunit protein of the coiled surface structure.
  • the name "curlin” is proposed for this subunit protein, curli for the structure and csga for the structural gene.
  • the curlin subunit appears to be strictly different from R coH pilins and coH pilins have several features in common such as cleavable signal peptide, two cysteine in the amino-terminal half, and several conserved amino acids in the amino and carboxy terminal regions thought to be involved in the subunit interactions. Likewise no homologoes were found with the N-methyl Phe class of pilus expressed by Neisseria gonorrhoeae and many other Gram negative species. Flagellin is not a true secretary protein since it is
  • Sequence analysis of a CsCl 2 -purified double-stranded plasmid DNA from pCSG4 was performed by denaturing approximately 4 ug of DNA with 2 M NaOH/2 mM EDTA and neutralizing it with 7.5 M ammonium acetate (pH 5). Appropriate oligonucleotides (1 pmole) were annealed to alkalidenatured DNA and sequenced using the Sequenase R protocol as described by the manufacturer (United States Biochemical, Cleveland, OH). Electrophoresis was performed in a 90 mM TEE buffer system for 2-5 hra at 45 mA in 8 M urea/6% polyacrylamide gels. Gels were fixed, dried, and exposed to Hyperfilm (Amersham).
  • R coli HB101 has been used as a host to clone and express a number of R coli pili types. In no other case it has been possible to obtain surface located pili by only expressing the pilin gene. All other known gene cluster
  • SUBSTITUTE SHEt i that have been examined today contain additional genes required for transport and assembly of the pilus fiber.
  • plasmid pFnb20 was present in R coli AA10 curli pili were not observed at 26°C as shown in FIG. 7 and the cells did not bind fibronectin.
  • Most R coli strains carry in their chromosome, genes for type 1 pili. Both R coli HblOl and AA10 are unable to form such pili. In the former strain some genes from the type 1 operon are still functioning whereas the entire type 1 gene cluster is deleted in AA10.
  • Plasmid pSJH9 carries all accessory genes required for type 1 pilus formation but is deleted for the fimA gene encoding the major pilin subunit.
  • AA10 harboring plasmid pSJH9 and pFnb20 did not bind fibronectin.
  • the ability of pFnb20 to express fibronectin binding and curli pili in HB101 does not seem to depend on the comnlementation from chromosomal type 1 pilus genes.
  • accessory genes located close to fnba could confer fibronectin binding to R c ⁇ ji AA10 this strain was transformed with the original cosmid clone pAO450.
  • strain AA10/pAO450 was not expressing fibronectin binding.
  • Minicell experiments revealed that strain AAIO is able to synthesize the FnbA protein at 26 °C as well as at 37° C. It is therefore concluded that the FnbA protein cannot be expressed for surface localization, nor be assembled into curli pili in this strain.
  • Escherichia coli is a common cause of bovine mastitis.
  • R. coli milk isolates from cases of acute mastitis do not differ from the normal faecal R coli flora in the cow.
  • the ability to bind to fibronectin stands out as a common property shared by 50-80% of the isolates studied.
  • the fnbA gene was cloned and characterized.
  • the fnb gene codes for a 17,000 dalton protein that is able to polymerize into curli pili-like structures when expressed in R. coH HblOl.
  • R coli pili are encoded from an operon consisting of 11 pap genes.
  • SUBSTITUTE SHEET outer membrane pore protein, and papD expressing a 28 kD periplasmic transport protein are needed in addition to pap A, the major subunit gene to form surface located pili. Even though other classes of pill adhesin genecluster appear to be less complex and contain fewer genes than the pap system all carry genes functionally equivalent to papC and papD.
  • R coH HB101 express the accessory proteins required for the biogenesis of curli Fnb pili. It is known that R coH HB101 carry some DNA that hybridize to cloned type 1 DNA. However, R coU HB101 do not express type 1 pili if transformed with a type 1 clone deleted for either the fimD (the papC equivalent) or the fimC (the papD equivalent) gene. It is therefore unlikely that it is chromosomal type 1 DNA that encodes the accessory proteins required for the formation of curli Fnb pili.
  • Curli Fnb pili are formed in HB101 at 26°C but not at 37°C.
  • the Fnb protein is synthesized to the same extent at both temperatures. It is thus suggested that the temperature regulation of piliation and fibronectin binding do not operate at the level of transcription but at the level of pilus biogenesis.
  • Pap pili and many other virulence associated properties are also temperature regulated but the effect of incubation tempera ⁇ ture is the reverse of what has been observed concerning the Fnb pili. In these former system transcription is increased with an increased temperature.
  • SUBSTITUTE SHEET This kind of temperature regulation is thought to reflect the adaptation of the micro-organism to the mammalian host.
  • Wound pathogens such as Staphylococcus aureus and Staphylococcus genera frequently bind to fibronectin.
  • the temperature of the skin is lower than 37°C. If fibronectin binding of R coH also reflects an adaptation to bind to wounds on the exterior of the host it would be an advantage for the micro ⁇ organism to have optimal binding at a temperature lower than 37°C.
  • Fibronectin binding curli pili actually plays a role in the pathogenesis of bovine mastitis.
  • the frequent presence of ulcers, due for example to machine milking on the udder might, however, enable colonization of fibronectin binding I coli.
  • Fibronectin is a large glycoprotein known to bind to a large set of proteins. It is therefore uncertain how specific the interaction is between fibronectin and Fnb pili.
  • the fnbA cosmid pAO450 was the single one out of 560 cosmids tested that expressed fibronectin binding in HB101. It is thus believed that this gene is the sole determinant of fibronectin binding in R. c ji AO12.
  • Strain A012 is a bovine faecal isolate obtained from a healthy cow.
  • Bacteria were normally grown in L-broth (Bertani 1951).
  • For the fibronectin binding assay bacteria were grown on CFA-agar (Evans, Inf. and Imm.. 21, 738-748) and containing 0.005% magnesium sulphate and 0-0005% magnesium chloride in 2% Bacto agar (Difco).
  • Competent cells for transformation were prepared with 50 mM CaCl 2 (Gene 6, 23-28, Dagert).
  • the antibiotics ampicillin (100 / ug/ml), kanamycin (50 / ug/ml) and chloramphenicol (20 / ug/ml) were used for selection of plasmid-containing strains. Unless otherwise stated, incubation of bacterial cultures was carried out at 37°C.
  • the present fibronectin binding protein will sometimes be synthesized in the cell wall and is not expressed as a pili. This protein thus synthesized can be isolated as well by known biochemical methods, such as affinity chromatography.
  • SUBSTITUTE SHEET fragments were subcloned into M13mplB and M13mpl9 vector (Messing, J and Nieira, J. Gene.. 19, 269-272 (1982)), and sequenced using the dideoxy chain terminating method of Sanger et al, P ⁇ AS. 74, 5463-5467.
  • D ⁇ A sequencing the bacteriophage T7 D ⁇ A polymerase, and SequenaseTM were used.
  • the primer used was Universal M13 17mer and synthesized 20mer oligonucleotides supplied by Symbicom, Umea, Sweden.
  • D ⁇ A was isolated as described by Lund et al. Plasmid D ⁇ A from clones carrying recombinant D ⁇ A was isolated by the alkaline lysis procedure (Maniatis, CSH, ⁇ .Y., USA).
  • Chromosomal DNA purified from R coH A012 were partially cleaved with endonuclease Sau3AI.
  • the DNA was size fractionated on a 10-40% linear sucrose gradient. Fractions containing DNA fragments larger than 20 kb in size were pooled and ligated into the BamHI site of the cosmid vector pJB8 as described by Maniatis, CSH Lab, N.Y., USA. Recombinant molecules were packaged in vitro into particles using a lambda DNA in vitro packaging kit (code N.334, Amersham). The phage was then used to infect R. coU HB101 by selecting for ampicillin resistant clones after growth on CFA agar plates at 30°C for 40-48 hrs.
  • Plasmid pFnbOl was constructed by subcloning a 4.9 kb large Sail fragment from this cosmid into pACYC184. An internal 3 kb large Sphl r Kpnl from pFnbOl was cloned into pUC18 (Messing) giving pFnblO. A number of subclones from pFnblO were generated by cloning into the polylinker site in pUC18. Plasmid pFnb59 is a Clal cutback derivative of
  • pFnblO SUBSTITUTE SHEET pFnblO.
  • Subclones were constructed as follows.
  • pFnblO consists of the 3.0 kb Kpnl-Pstl fragment of the original clone ligated into the polylinker cloning cassette of pUC18, while pFnb3O is a Clal deletion derivative of pFnblO.
  • Plasmid pFnb20 was obtained by cloning the 1.5 kb Sphl-B lll fragment from the original plasmid into pUCl ⁇ . Analysis of this construct which lacks the 5' terminal end of the gene coding for the 49 kD peptide showed that this protein was not necessary for curli production or for fibronectin binding.
  • Plasmids pFnb56 and pFnb46 are SphlHpal and Clal-Bgi ⁇ subclones, respectively of pFnblO in pUC18. Plasmid pFnb59 was obtained by first subcloning the Kpnl-Bgi ⁇ fragment of the original plasmid in pUC18 and then inserting the aminoglycoside-3'-phosphotransf erase gene (APH) from the mobilization plasmid pUC4K into the Clal ! site. To perform the fibronectin binding assay bacteria were grown an CFA-agar plates for 42-48 hrs at 26°C or 37°C.
  • APH aminoglycoside-3'-phosphotransf erase gene
  • Accl digested plasmid pUC-4K (Pharmacia, Uppsala, Sweden) carrying the kanamycin resistance gene from transposon Tn903 coding for aminoglycoside 3 phosphotransferase (APH) was ligated to Clal digested pFnb56.
  • Transformants in HB101 were screened for ampicillin and kanamycin resistance.
  • One such clone carrying the Kana R fragment at the Clal site was denoted pFnb59.
  • the fibronectin binding assay was a modification of the procedure described by G. Froman et al, (JBC, 259, 14899-14905). Bacteria were inoculated on CFA plates for 42-48 hrs at 26°C or 37°C. Cells were resuspended in cold phosphate-buffered saline (pH 7.5) to an optimal density of 10 9 cfu/ml. 100 ul of cells were added to an assay tube containing 1 ml of PBS + 0-1 % Tween 80 + 100 /ul of 125 I-fibronectin (5x10 s cpm) and the mixture was end over incubated in room temperature for 1 hr. Tubes were centrifuged in Eppendorf centrifuge for five min. Supematants were carefully
  • Plasmid constructs were transformed into the minicell-producing strain AAIO. Preparation and labelling of minicells with 35 S-methionine were as described by Thompson and Achtman (Mol. Gen. Genet. 165. 295-304 (1978)).
  • radioactive samples were separated on linear 15 % (wt vol) SDS- polyacrylamide gels (Laemmli, UK, Nature. 227. 880-885, (1970)). The gels were fixed, stained, destained, and exposed to X-ray film (DuPont) for 1-5 days. Molecular weight standards were from Pharmacia Fine Chemicals, Uppsala, Sweden.
  • Precursor form of proteins encoded by different constructions were monitored after radiolabelling of minicells in the presence of 9% ethanol (Palva, J. Bact.. 146. 325-330) and analyzed on SDS-polyacrylamide gels.
  • Electron microscopy was performed using a JEOL 100B microscope with 100-mesh copper grids coated with thin films of 2% Formvar. Bacteria from CFA agar plates were resuspended in 10 mM Tris-HCl,-pH 7.5 + 10 mM MgCl 2 , and placed on the grid. Grids were washed with buffer and negatively stained for 5 sec. with 3.55% ammonium molybdate, followed by washing with redistilled water.
  • the present fibronectin binding protein can be used for immunization, whereby the protein, preferably in combination with a fusion protein to create a large antigen to respond to, or the pili shaken off from the R coH expressing the curli pili when grown around about 30 °C, preferably at 26°C, or the inactivated R coli comprising the curli pili consisting of the fibronectin binding 17 kD protein, is injected in dosages causing immunological reaction in the host mammal.
  • the fibronectin preferably in combination with a fusion protein to create a large antigen to respond to, or the pili shaken off from the R coH expressing the curli pili when grown around about 30 °C, preferably at 26°C, or the inactivated R coli comprising the curli pili consisting of the fibronectin binding 17 kD protein
  • SUBSTITUTE SHEET binding protein can be used in vaccination of ruminants against mastitis caused by R coH infections.
  • the fibronectin binding protein can further be used for immunization against urinary tract infections, normally caused by R coH, or intestinal infections normally caused by R cpji, such as ulcerous colitis.
  • the fibronectin binding protein of this invention has shown to form antibodies against R cjp ⁇ related infections.
  • the fibronectin binding protein can be used to block an infection in an open skin wound by wound treatment using the fibronectin binding protein in a suspension.
  • the fibronectin binding protein can be used for the treatment of wounds, e.g., for blocking protein receptors, or for immunization (vaccination).
  • the host body produces specific antibodies which can protect against invasion of bacterial strains comprising such a fibronectin binding protein.
  • the antibodies block the adherence of the bacterial strains to damaged tissue.
  • colonizing of tissue damage examples include: a) colonizing of wounds in skin and connective tissue, which wounds have been caused by a mechanical trauma, chemical damage, and/or thermical damage; b) colonizing of wounds on mucous membranes such as in the mouth cavity, or in the mammary glands, urethra, or vagina; c) colonizing on connective tissue proteins, which have been ex ⁇ posed by minimal tissue damage (micro lesion) in connection with epithelium and endothelium (mastitis, heart valve infection, hip exchange surgery).
  • minimal tissue damage micro lesion
  • the protein, or polypeptide, or curli pili, or whole inactivated bacteria is dispersed in sterile, isotonic saline solution, optionally while adding a pharmaceutically acceptable dispersing agent.
  • sterile, isotonic saline solution optionally while adding a pharmaceutically acceptable dispersing agent.
  • adjuvants can further be used in order to sustain the release in the tissue, and thus expose the protein for a longer time to the immuno defense system of
  • a suitable dosage to obtain immunization is 0.5 to 5 ug of fnb protein per kg bodyweight and injection of immunization.
  • vaccinations should be carried out at more than one consecutive occasions with an interval of 1 to 3 weeks, preferably at three occasions.
  • the protein When using the present fnb protein for topical local administration the protein is dispersed in an isotonic saline solution to a concentration of 25 to 250 u per ml. The wounds are then treated with such an amount only to obtain a complete wetting of the wound surface. For an average wound thus only a couple of milliliters of solution are used in this way. After treatment using the protein solution the wounds are suitably washed with isotonic saline or another suitable wound treatment solution.
  • fibronectin binding protein, or the synthesized polypeptide of the present invention can be used to diagnose bacterial infections caused by R. coU strains, whereby a fibronectin binding protein of the present invention is immobilized on a solid carrier, such as small latex or Sepharose R beads, whereupon sera containing antibodies are allowed to pass and react with the fibronectin binding protein thus immobilized. The agglutination is then measured by known methods.
  • a solid carrier such as small latex or Sepharose R beads
  • fibronectin binding protein or the polypeptide can be used in ELISA test (E. Engvall, "Enzyme Linked Immuno Sorbent Assay," Med. Biol. 55, 193, (1977)).
  • ELISA test E. Engvall, "Enzyme Linked Immuno Sorbent Assay," Med. Biol. 55, 193, (1977)
  • wells in a polystyrene microtitre plates are coated with the fnb protein and incubated over night at 4°C. The plates are then thoroughly washed using PBS containing 0.05% Tween 20, and dried. Serial dilution of the patient serum is made in PBS-Tween, are added to the wells, and incubated at 30°C for 1.5 hrs.
  • antihuman-IgG conjugated with an enzyme or a horseradish peroxidase, or an alkaline phosphatase is added to the wells and incubated at 300°C for 1.5 hrs., whereupon when the IgG has been bound thereto, and after rinsing, an enzyme, or a horseradish peroxidase, or an alkaline phosphatase is added to the wells and incubated at 300°C for 1.5 hrs., whereupon when the IgG has been bound thereto, and after rinsing, an enzyme, or a horseradish peroxidase, or an alkaline phosphatase is added to the wells and incubated at 300°C for 1.5 hrs., whereupon when the IgG has been bound thereto, and after rinsing, an enzyme, or a horseradish peroxidase, or an alkaline phosphatase is added to the wells and incubated at 300°C for 1.5 hrs.,
  • SUBSTITUTE SHEET enzyme substrate is added, a p-nitrophosphate in case of an alkaline phosphatase, or orthophenylene diamine substrate (OPD) in case a peroxidase has been used, respectively.
  • the plates comprising the wells are thus then rinsed using a citrate buffer containing 0.05S% OPD, and 0,005% H 2 O 2 , and incubated at 30°C for 10 min. Enzyme reaction is stopped by adding a 4N solution of H 2 SO 4 to each well. The color development is measured using a spectrophotometer.
  • fibronectin binding protein includes any of the polypeptide sequences as well, which polypeptide sequences form the minimal fibronectin binding site of the complete protein.
  • Figure legends Figure 1 Physical map of the different pFnb plasmid constructs. Fnb-f- or - indicate fibronectin binding phenotype. Constructs: pFnblO SphI r KpnI fragment in pUC18
  • Figure 2 Restriction map and gene organization of fnb gene. The hatched boxes indicate the 17 kD.
  • Electron microscopy was performed with a JOEL 100B microscope with 100-mesh copper grids coated with thin films of 2% Formvar. Bacteria from CFA-agar plates were resuspended in lOmM tris-HCl, pH 7,5-lOmM MgCl 2 and placed on the grid. Grids were washed with buffer and negatively stained for 5 sec. with 3.55% ammonium molybate, followed by washing with redistilled water.

Abstract

La présente invention se rapporte à une nouvelle protéine de liaison de la fibronectine à partir d'E. coli se présentant sous la forme de curli pili, une nouvelle molécule d'ADN hybride recombinée comprenant une séquence nucléotidique à partir d'E. coli codant pour une protéine ou un polypeptide ayant des propriétés de liaison de la fibronectine.
PCT/US1993/010547 1992-11-03 1993-11-03 Proteine de liaison de la fibronectine et sa preparation WO1994010330A1 (fr)

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Application Number Priority Date Filing Date Title
AU55907/94A AU692140B2 (en) 1992-11-03 1993-11-03 Fibronectin binding protein as well as its preparation
NO951679A NO951679L (no) 1992-11-03 1995-05-02 Fibronektin-bindende protein, samt fremstilling derav
FI952108A FI952108A (fi) 1992-11-03 1995-05-03 Fibronnektiiniä sitova proteiini sekä sen valmistaminen

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US97084692A 1992-11-03 1992-11-03
US07/970,846 1992-11-03

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US6685943B1 (en) 1997-01-21 2004-02-03 The Texas A&M University System Fibronectin binding protein compositions and methods of use
ATE342990T1 (de) 2001-08-10 2006-11-15 Bioleaders Corp Oberflächenexpressionsvektoren mit pgsbca, dem für poly-gamma-glutamat-synthetase codierenden gen, sowie verfahren zur expression eines zielproteins an der oberfläche eines den vektor benutzenden mikroorganismus
GB0202275D0 (en) * 2002-01-31 2002-03-20 Hansa Medica Ab Peptide
KR20200000972A (ko) 2018-06-26 2020-01-06 주식회사 바이오리더스 바실러스 속 균주 유래의 폴리감마글루탐산 합성유전자를 이용한 표면발현벡터 및 이를 이용한 단백질의 미생물표면 발현 방법
JP7147060B2 (ja) 2018-10-10 2022-10-04 バイオリーダース コーポレイション ラクトバチルス・カゼイ由来ガラクトース・ムタロターゼ遺伝子のプロモーターを用いた常時的高発現表面発現ベクターおよびその利用

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