WO1994010330A1 - Proteine de liaison de la fibronectine et sa preparation - Google Patents

Proteine de liaison de la fibronectine et sa preparation Download PDF

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WO1994010330A1
WO1994010330A1 PCT/US1993/010547 US9310547W WO9410330A1 WO 1994010330 A1 WO1994010330 A1 WO 1994010330A1 US 9310547 W US9310547 W US 9310547W WO 9410330 A1 WO9410330 A1 WO 9410330A1
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protein
fibronectin binding
coli
binding protein
polypeptide
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WO1994010330A9 (fr
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Staffan Normark
Arne Olsen
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REA TERESA STANEK
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REA TERESA STANEK
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Priority to AU55907/94A priority Critical patent/AU692140B2/en
Priority to FI952108A priority patent/FI952108L/fi
Publication of WO1994010330A1 publication Critical patent/WO1994010330A1/fr
Publication of WO1994010330A9 publication Critical patent/WO1994010330A9/fr
Priority to NO951679A priority patent/NO951679L/no
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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 microorganims 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-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. 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 fulfil a critical role in clearance by phagocytes of autologous tissue debris, immune complexes, and bacteria. Fibronectins also 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.
  • E. coli can express a variety of adhesins with differing binding specificities. The majority of these adhesins recognize carbohydrate moities present on glycoconjugates. E. coli may also express binding to matrix proteins such as fibronectin, laminin, and collagen.
  • Uropatogenic E. coli expressing the 075 ⁇ adhesin bind tubular basement membranes and to the Bowman capsule known to be rich in laminin. The purified 075 ⁇ adhesin was specifically found to bind laminin.
  • E. coli isolated from patients with ulcerous colitis frequently bind matrix proteins.
  • E. coli isolates from bovine mastitis have been shown to bind to fibronectin at a high frequency. Below a native fibronectin binding protein from E. coli is disclosed, as well as the cloning of the fibronectin binding, fnbA gene from a bovine isolate of E. coli that express curli pili and fibronectin binding when present in E. coli HB101.
  • Chemically fibronectin is a large glycoprotein (M 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.
  • 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 micro- organism E. coli HB101/pFnb20 encompassing the plasmid encoding for said nucleotide sequence was deposited May 05, 1988 at Deut- sche Sammlung von Mikroorganismen und Zellkulturen, Braun- schweig, FRG under the deposition number DSM 4585.
  • 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 E. coli HB101. Strain AO12 expressed fibronectin binding when grown on CFA plates at 30°C, but to a lesser extent at 37oC. Transductants were thus screened for fibronectin binding after growth on CFA plates for 40 hrs at 30oC. Out of 560 transductants one, AO450, showed fibronectin binding. The recombinant cosmid in this transductant, designated pAO450, was isolated and shown to contain an insert of about 24 kb.
  • pAO450 was digested with restriction endonuclease Sail and subcloned into the vector pACYC184, giving pFnb01.
  • the subclone pFnb10 was digested with a series of restriction endonucleases to prepare a restriction map as shown in FIG. 1.
  • To further localize the region on pFnb10 expressing fibronectin binding several subclones were constructed from pFnb10 as were various deletion derivatives. These constructs were tested for their ability to confer binding to E. coli HB101.
  • the HpaI and SphI sites delineates the 1.2 kb region required for fibronectin binding as shown in FIG. 1.
  • Plasmid encoded polypeptides shown in FIG. 1 were transformed into the minicell producing strain AA10. Plasmid encoded polypeptides were analysed from S-methionine labelled minicells. Plasmid pFnb10 expressed two polypeptides with molecular masses of 43 kD and 17 kD respectively as shown in FIG 2. pFnb30 is a deletion derivative of pFnb10 that lacks a 0.9 kb ClaI fragment. This derivative no longer confer fibronectin binding to E. 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 ClaI 2 and KpnI sites on pFnb10, since the region between SphI 1 and ClaI 1 site is to small (0.4 kb) to encompass a gene coding for a 49 kD protein. It is also concluded that the 49 kD polypeptide is not required for fibronectin binding. Plasmids pFnb20 and pFnb58 both expressed fibronectin binding in E. coli The 17 kD polypeptide but not the 49 kD polypeptide was encoded by pFnb20 showing that the gene for the latter polypeptide must span the SphI 2 site.
  • the HpaI-SphI 1 subclone pFnb56 no longer expressed the 17 kD polypeptide. Instead a slightly larger polypeptide species of 19 kD was expressed. An 1.5 kb large fragment carrying the aminoglycoside 3 phosphotransferase gene plasmid of pUC-4K was cloned into the single ClaI site of pFnb56 giving pFnb59. This derivative no longer mediated fibronectin binding. In minicells there was no expression of the 19 kD polypeptide.
  • the 19 kD polypeptide is due the deletion in the pFnb56 of the normal translational stop codon of the gene for the 17 kD polypeptide giving rise to a slightly longer translational product containing some carboxyterminal amino acids encoded by the vector.
  • the coiled surface structure could be shared from the surface of 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 Immobulon filter and the aminoterminal sequence determined by sequential Edman degradation .
  • the iso- lated protein was the same 17 kD gene product of the structural gene spanning the ClaI, and the HpaI sites in pFnb20 this region was sequenced.
  • One open reading frame consisting of 133 codons was identified spanning the ClaI 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 E. coli pilins and E. coli pilins have several features in commnon such 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-subunit interactions.
  • cleavable signal peptide two cysteine in the amino-terminal half
  • several conserved amino acids in the amino- and carboxy terminal regions thought to be involved in the subunit-subunit interactions.
  • no homologoes were found with the N-methyl Phe class of pilus expressed by Neisseria qonorrhoeae and many other Gram negative species.
  • Flagellin is not a true secretory protein since it is transported directly from the cy- toplasm to the growing tip through the hollow flagellum.
  • the small diameter of the curli suggest that they are assembled from the base.
  • fnbA E. coli pilin proteins are all known to be produced in a precursor form with a cleavable N-terminal signalpeptide.
  • Fnb protein was synthetised from fnbA in a precursor form, minicells expressing Fnb from plasmids were treated with 9% ethanol to prevent processing. There was no appearance of a large molecular weight species suggesting that Fnb is not synthetized with a cleavable signalpeptide.
  • DNA from pCSG4 was performed by denaturring 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
  • Electrophoresis was performed in a 90 mM TBE buffer system for 2-5 hrs at 45 mA in 8 M urea/6% polyacrylamide gels. Gels were fixed, dried, and exposed to Hyperfilm (Amersham).
  • E. coli strain AO12 grown at 26°C expressed unusually curli pili like structures when viewed under the electron microscope as shown in FIG. 4. No such structures can be seen on E. coli HB101 growing at either 26°C or 37°C.
  • cells of HB101 harbouring either pFnb20 of pFnb56 produced large amounts of curli pili structures when grown on CFA medium at 26oC as shown in FIG. 5.
  • no surface structures was found on HB101/pFnb20 and HB101/pFnb56 after growth at 37°C as shown in FIG. 6, a temperature at which these strains do not express fibronectin binding.
  • curli pili in HB101 was strictly correlated to the expression of the Fnb protein. Moreover, the expression of curli pili correlated strictly with the ability of HB101 to bind to fibronectin.
  • E. coli HB101 has been used as a host to clone and express a number of E. 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 that have been examined today contain additional genes required for transport and assembly of the pilus fiber. When plasmid pFnb20 was present in E_. coli AA10 curli pili were not observed at 26°C as shown in FIG. 7 and the cells did not bind fibronectin. Most E. coli strains carry in their chromosome, genes for type 1 pili. Both E. coli Hb101 and AA10 are unable to form such pili.
  • Eschericia coli is a common cause of bovine mastitis.
  • E. coli milk isolates from cases of acute mastitis do not differ from the normal faecal E. 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 E. coli Hb101.
  • E. coli pili are encoded from an operon consisting of 11 pap genes.
  • the accessory genes papC coding for an 88 kD outer membrane pore protein, and papD expressing a 28 kD periplasmic transport protein arc needed in addition to papA, the major subunit gene to form surface located pili.
  • papC coding for an 88 kD outer membrane pore protein
  • papD expressing a 28 kD periplasmic transport protein
  • E. coli HB101 express the accessory proteins required for the biogenesis of curli Fnb pili. It is known that E. coli HB101 carry some DNA that hybridize to cloned type 1 DNA. However, E. coli 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 (tht 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 temperature is the reverse of what has been observed concerning the Fnb pili. In these former system transcription is increased with an increased temperature. 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 E. coli 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 E. 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 E. coli A012.
  • Strain A012 is a bovine faecal isolate obtained from a healthy cow.
  • E. coli K12 strains were tested and analysed for the production of curli and the ability to bind fibronectin.
  • Strain C600 thr-1 leuB6 lacY1 suoE44 rfbD1 thi-1 tonA21
  • the proA2 deletion mutant AB1157 did not, as expected express neither property.
  • the minicell producing E. coli K12 strain AA10 carries the crl region as reveal ed by DNA-DNA hybridization using the 1.5 kb large BglII-SphI fragment as a probe, but did not express curli and did not bind soluble fibronectin.
  • 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 37oC.
  • 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.
  • the primer used was Universal M13 17mer and synthesized 20mer oligonucleotides supplied by Symbicom, Umea, Sweden. Isolation of chromosomal and plasmid DNA.
  • Plasmid DNA from clones carrying recombinant DNA was isolated by the alkaline lysis procedure (Maniatis, CSH, N.Y., USA).
  • Chromosomal DNA purified from E. coli AO12 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 E. coli HB101 by selecting for ampicillin resistant clones after growth on CFA agar plates at 30°C for 40-48 hrs.
  • Plasmid pFnb01 was constructed by subcloning a 4.9 kb large SalI fragment from this cosmid into pACYC184. An internal 3 kb large SphI 1 -KpnI from pFnb01 was cloned into pUC18 (Messing) giving pFnb10. A number of subclones from pFnb10 were generated by cloning into the polylinker site in pUC18. Plasmid pFnb59 is a ClaI cut back derivative of pFnb10. Subclones were constructed as follows.
  • pFnb10 consists of the 3.0 kb KpnI-PstI fragment of the original clone ligated into the polylinker cloning cassette of pUC18, while pFnb30 is a Clal deletion derivative of pFnb10. Plasmid pFnb20 was obtained bby cloning the 1.5 kb Sphl-BglII fragment from the original plasmid into pUC18.
  • Plasmids pFnb56 and pFnb46 are SphlHpal and ClaI-BglII subclones, respectively of pFnb10 in pUC18. Plasmid pFnb59 was obtained by first subcloning the Kpnl-BglII fragment of the origi nal plasmid in pUC18 and then inserting the aminoglycoside-3'- phosphotransferase gene (APH) from the mobilization plasmid pUC- 4K into the Clal 1 site. To perform the fibronectin binding assay bacteria waere gromn on CFA-agar plates for 42-48 hrs at 26oC or 37°C.
  • APH aminoglycoside-3'- phosphotransferase gene
  • AccI digested plasmid pUC-4K (Pharmacia, Uppsala, Sweden) carrying the kanamycin resistance gene from transposon Tn903 coding for aminoglycoside 3 phosphotransferase (APH) was ligated to ClaI digested pFnb56.
  • Transformants in HB101 were screened for ampicillin and kanamycin resistance.
  • One such clone carrying the Kana fragment at the ClaI 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 26oC or
  • Plasmid constructs were transformed into the minicell-producing strain AA10. Preparation and labelling of minicells with 35 S-methionine were as described by Thompson and Achtman (Mol. Gen.
  • the radioactive samples were separated on linear 15% (wt/vol) SDS-polyacrylamide gels (Laemmli, UK, Nature, 227, 880-885,
  • 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 E.
  • the fibronectin binding protein can be used in vaccination of ruminants against mastitis caused by E. coli infections.
  • the fibronectin binding protein can further be used for immunization against urinary tract infections, normally caused by E. coli, or intestinal infections normally caused by E. coli, such as ulcerous colitis.
  • the fibronectin binding protein of this invention has shown to form antibodies against E.coli 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.
  • fnb protein or a synthesized amino acid polypeptide for the purpose of immunization (vaccination) in mammals, including humans, 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.
  • 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 time to the immuno defence system of a body.
  • the injectible solutions will usually be given subcutaneously or intramuscularly.
  • 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 millilitres 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 diagnoze bacterial infections caused by E. coli strains, whereby a fibronectin binding protein of the present invention is immobilized on a solid carrier, such as small latex or Sepharose 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.
  • fibronectin binding protein or the polypeptide can be used in ELISA test (Enzyme Linked Immuno Sorbent Assay; E Engvall, Med. Biol. 55, 193, (1977)).
  • ELISA test Enzyme Linked Immuno Sorbent Assay; E Engvall, Med. Biol. 55, 193, (1977)
  • wells in a polystyrene microtitre plate 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.
  • a fluoroscense measurement can be used as well.
  • Another method to diagnoze E. coli infections is by using the DNA gene probe method based on the fnb protein sequence or the synthetic polypeptide sequence.
  • a solid carrier such as a polystyrene plate as mentioned above, by e.g. adding a milk in the case of diagnozing a mastitis, to the surface.
  • the DNA gene probe optionally labelled enzymatically, or by a radioactive isotope is then added to the solid surface plate comprising the DNA sequence, whereby the DNA gene probe attaches to the sequence where appearing.
  • the enzyme or the radioactive isotope can then readily be determined by known methods.
  • fibronectin binding protein includes any of the polypeptide sequences as well, which polypeptide sequences form the minimal fibronectin binding site of the complete protein.
  • 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 10mM tris-HCl, pH 7,5-10mM 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.

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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 Ceased 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
FI952108A FI952108L (fi) 1992-11-03 1993-11-03 Fibronnektiiniä sitova proteiini sekä sen valmistaminen
NO951679A NO951679L (no) 1992-11-03 1995-05-02 Fibronektin-bindende protein, samt fremstilling derav

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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
WO2003064446A3 (fr) * 2002-01-31 2004-04-01 Hansa Medical Res Aktiebolag Peptides
US7553636B2 (en) 2001-08-10 2009-06-30 Bioleaders Corporation Surface expression vectors having pgsBCA the gene coding poly-gamma-glutamate synthetase, and a method for expression of target protein at the surface of microorganism using the vector
WO2020004877A1 (fr) 2018-06-26 2020-01-02 주식회사 바이오리더스 Vecteur d'expression de surface utilisant un gène synthétique de poly-gamma-glutamate dérivé d'une souche dans bacillus, et procédé d'expression de protéine sur la surface d'un micro-organisme l'utilisant
WO2020076078A1 (fr) 2018-10-10 2020-04-16 주식회사 바이오리더스 Vecteur d'expression de surface pour expression élevée constitutive à l'aide d'un promoteur de gène de galactose mutarotase dérivé de lactobacillus casei, et son utilisation

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SE8801723D0 (sv) * 1988-05-06 1988-05-06 Staffan Normark Fibronectin binding protein as well as its preparation

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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
US7553636B2 (en) 2001-08-10 2009-06-30 Bioleaders Corporation Surface expression vectors having pgsBCA the gene coding poly-gamma-glutamate synthetase, and a method for expression of target protein at the surface of microorganism using the vector
WO2003064446A3 (fr) * 2002-01-31 2004-04-01 Hansa Medical Res Aktiebolag Peptides
WO2020004877A1 (fr) 2018-06-26 2020-01-02 주식회사 바이오리더스 Vecteur d'expression de surface utilisant un gène synthétique de poly-gamma-glutamate dérivé d'une souche dans bacillus, et procédé d'expression de protéine sur la surface d'un micro-organisme l'utilisant
WO2020076078A1 (fr) 2018-10-10 2020-04-16 주식회사 바이오리더스 Vecteur d'expression de surface pour expression élevée constitutive à l'aide d'un promoteur de gène de galactose mutarotase dérivé de lactobacillus casei, et son utilisation

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NO951679D0 (no) 1995-05-02
FI952108A7 (fi) 1995-07-03
AU692140B2 (en) 1998-06-04
NO951679L (no) 1995-07-03
FI952108L (fi) 1995-07-03
AU5590794A (en) 1994-05-24
FI952108A0 (fi) 1995-05-03
CA2148479A1 (fr) 1994-05-11

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