Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to peptides for use in cell adhesion and wound healing. More particularly, the present invention relates to the use in cell adhesion and wound healing of peptides containing one or more copies of the 2 nd and/or 4 th fas-1 domain of ⁇ ig-h3, said 2 nd and 4 th domains sharing a high homology in two amino acids, aspartic acid and isoleucine, essential for binding to integrin and thus mediating cell adhesion. Also, the present invention is concerned with an expression system for the peptides useful in cell adhesion and wound healing.
• ⁇ ig-h.3 is an extracellular matrix protein whose expression is induced in various cell lines, including human melanoma cells, mammary ephithelial cells, keratinocytes, and lung fibroblasts, following signaling by active TGF- ⁇ (Skonier, J. et al., DNA Cell Biol. 13, 571, 1994).
• the ⁇ ig-h3 gene was first isolated by differential hybridization screening of a cDNA library made from a human lung adenocarcinoma cell line that had been treated with TGF- ⁇ .
• ⁇ ig-h3 gene encodes a 683-amino acid protein that is highly conserved between species.
• RGD Arg-Gly-Asp
• ⁇ ig-h3 is known to be involved in cell growth and proliferation, wound healing, and cell adhesion, although the underlying mechanisms for these functions are still unclear. However, ⁇ ig-h3 seems to play an important role in the morphogenesis and interactions with cells and extracellular matrix proteins in various tissues.
• ⁇ ig-h3 Some evidence related to the role of ⁇ ig-h3 in mediating cell attachment and detachment is provided by several studies. For example, purified ⁇ ig-h3 protein is found to promote the attachment and spreading of skin fibroblasts while inhibiting the adhesion of A549, HeLa and i-38 cells in serum-free media. Particularly, ⁇ ig-h3 is known to have inhibitory activity against tumor cell growth, and to affect colony formation and morphology. The inhibitory activity was demonstrated by the report in which transfection of ⁇ ig-h3 expression plasmids into CHO (Chinese hamster ovary) cells led to marked decreases in cell proliferation and the ability of these cells to form tumors in nude mice.
• CHO Choinese hamster ovary
• ⁇ ig-h3 a cell adhesion molecule induced by TGF- ⁇ in various cell lines, plays a very important role in cell growth, cell differentiation, wound healing, morphogenesis and cell adhesion (Rawe, I. M. et al., Invest. Ophthalmol. Vis. Sci. 38, 893, 1997; Lebaron, R. G. et al., J. Invest. Dermatol. 104, 844, 1995).
• ⁇ ig-h3 contains four 140 amino acid repeats with internal homology, namely fas-1 domains.
• the internal repeat domains have highly conserved sequences found in secretory proteins or membrane proteins of various species, including mammals, insects, sea urchins, plants, yeasts, and bacteria. Proteins containing the conserved sequence are exemplified by periostin, fasciclin I, sea urchin HLC-2, algal-CAM and mycobacterium MPB70.
• the conserved domain in these proteins (hereinafter referred to as "fas-1”) consists of about 110 to 140 amino acids with two highly conserved branches, HI and H2, of about 10 amino acids each (Kawamoto, T. et al., Biochem. Biophys. Acta.
• fas-1 domains are found in ⁇ ig-h3, periostin, and fasciclin I, two fas-1 domains in HLC-2, and only one fas-1 domain in MPB70.
• ⁇ ig-h3, periostin, and fasciclin 1 are reported to mediate the adhesion of fibroblasts, osteoblasts, and nerve cells, respectively.
• algal-CAM is a cell adhesion molecule present in embryos of the algae Volvox (LeBaron, R. G., et al., J. Invest. Dermatol. 104, 844, 1995; Horiuchi, K. et al., J. Bone Miner. Res. 14, 1239, 1999; Huber, 0. et al . , EMBO J. 13, 4212, 1994) .
• ⁇ ig-h3 promotes the spreading of fibroblasts via integrin ⁇ l ⁇ l whereas the RGD motif of ⁇ ig- h3 is not necessary for mediating the cell adhesion property of ⁇ ig-h3.
• ⁇ ig-h3 binds specifically to integrin to enhance the cell adhesion and spreading of cells irrespective of RGD motif (Ohno, S. et al., Bioch . Biophys. Acta 1451, 196, 1999).
• the conserved peptides Hi and H2 of ⁇ ig-h3 were found to have no influence on ⁇ ig-h3-mediated cell adhesion.
• wound healing is a tissue response to trauma, leading to tissue repair through complex biological processes, including chemotaxis, cell differentiation and replication, matrix protein synthesis, angiogenesis, and wound reconstitution (Steed, D. L., et al . , Clin. Plast. Surg. 25, 397, 1998).
• Growth factors are representative materials that appear in the early stage of the wound healing process and control the subsequent wound healing process. Having strong influence over all stages of wound healing, growth factors act to control the growth, differentiation and metabolism of cells and reorganize the environs of the wound by their chemotactic properties which attract various cells types that are involved in inflammation and tissue repair, cellular proliferation, stimulating angiogenesis and the synthesis and degradation of the extracellular matrix.
• PDGF vascular endothelial growth factor
• TGF- ⁇ transforming growth factor-beta
• EGF epidermal growth factor
• bFGF basic fibroblast growth factor
• IGF insulin-like growth factor
• VEGF vascular endothelial growth factor
• TGF- ⁇ is the most representative.
• TGF- ⁇ l the cytokine plays important roles in the growth and differentiation of various cells and has various complex functions, including control of cell growth, regulation of immune responses, stimulation of osteogenesis, induction of cartilage specific macromolecules, and promotion of wound healing (Bennett, N. T. et al . , Am. J. Surg. 165, 728, 1993).
• Appearing in the ephithelium during wound healing, TGF- ⁇ is believed to stimulate the expression of integrin within keratinocytes during re-epithelialization.
• TGF- ⁇ 3 mRNA is expressed in the epithelia of normal skin and acute and chronic wounds, while TGF- ⁇ l mRNA is not expressed in normal skin and chronic wounds, but expressed in the epithelial layer regenerated from acute wounds, and nowhere is expressed TGF- ⁇ 2 mRNA (Schmid, P. et al., J. Pathol . 171, 191, 1993) . Based on the effects, even though their mechanisms are not firmly established, TGF- ⁇ is expected to play a major role in re-epithelialization.
• ⁇ ig-h3 is up-regulated by TGF- ⁇ , suggesting that ⁇ ig-h3 is involved in the mediation of some signals of TGF- ⁇ .
• CHO (Chinese hamster ovary) cells transformed with ⁇ ig-h3 expression plasmids are reported to show decreased tumorigenic ability (Skonier, J. et al . , DNA Cell Biol. 13, 571, 1994).
• ⁇ ig-h3 expression is down-regulated in dexamethasone-treated stem cells, some tumor cells and the fibroblasts cultured from the skin lesion sites afflicted with localized hyperostosis of melorheostosis.
• ⁇ ig-h3 is also reported to serve as a negative regulator of osteogenesis (Genini, M. et al., Int. J. Cancer 66, 571, 1996; Schenker, T. et al . , Exp. Cell. Res. 239, 161, 1998; Kim, J. et al . , J. Cell Biochem. 77, 169, 2000) .
• ⁇ ig-h3 known as a cell adhesion molecule, promotes the adhesion and spreading of fibroblasts in the dermis.
• ⁇ ig-h3 According to studies into the distribution of ⁇ ig-h3 in eye tissues, it is reported that the adhesion molecule is expressed in corneal epithelia of normal adults, intracorneal fetal stromal cells, and the endothelial and stromal cells in the process of wound healing. In addition, ⁇ ig-h3 is expressed in the j uxtaglo erular apparatus and proximal tubules of the kidneys, and its expression is increased in diabetes mellitus. Further, it is found in subendothelial smooth muscles of the coronary arteries of normal persons, and its amount is increased in the endometria of blood vessels in the case of arteriosclerosis.
• recombinant proteins which were designed to have the 2 nd and/or 4 th fas-1 domain of ⁇ ig-h3 were identified as being identical to wild type ⁇ ig-h.3 in cell attachment and spreading activity and wound healing effect.
• a recombinant protein comprising a portion of domains of ⁇ ig-h3, useful in mammalian cell attachment .
• expression vectors p ⁇ ig-h3 D- II, p ⁇ ig-h3 D-IV, and p ⁇ ig-h3 D-IV 4X capable of expressing the 2 nd and 4 th fas-1 domain of ⁇ ig-h3 corresponding to amino acids 237-377 and 498-637, respectively.
• novel E. coli strains transformed with the expression vectors p ⁇ ig-h3 D-II, p ⁇ ig- h3 D-IV, and p ⁇ ig-h3 D-IV 4X, identified as E. coli BL21/His ⁇ -g (accession No. KCTC 0905BP) , E. coli BL21/His ⁇ -e
• a method for attaching cells comprising the steps of: preparing a recombinant protein containing one or more copies of the 2 nd and/or 4 th domain of ⁇ ig-h3, by use of an expression vector; coating the recombinant protein onto a solid support; and applying cells to the protein-coated solid support.
• the use of the recombinant protein in cell attachment comprising the steps of: preparing a recombinant protein containing one or more copies of the 2 nd and/or 4 th domain of ⁇ ig-h3, by use of an expression vector; coating the recombinant protein onto a solid support; and applying cells to the protein-coated solid support.
• a method for healing wounds comprising the steps of: coating a solid support with a recombinant protein containing one or more copies of the 2 nd and/or the 4 th domain of ⁇ ig-h3; attaching skin cells to the solid support; and applying the solid support to wounds .
• Fig. la is a schematic diagram showing recombinant proteins ⁇ igh3- T and ⁇ igh3- ⁇ RGD, wherein conserved regions are represented by 0 and H, and RGD motif by ®.
• Fig. 2 is a photograph showing SDS-PAGE results of recombinant proteins J3igh3-WT and ⁇ igh3- ⁇ RGD.
• Fig. 3 is a microphotograph showing HCE cell adhesion and spreading effects of recombinant proteins ⁇ igh3-WT and ⁇ igh3- ⁇ RGD after dying with crystal violet.
• Fig. 4 shows curves in which the HCE cell adhesion and spreading activities of the recombinant proteins ⁇ igh3-WT and ⁇ igh3- ⁇ RGD are found to be concentration-dependent as measured by the count (A) and surface area (B) of attached cells .
• Fig. 5 shows histograms in which the HCE cell adhesion activities of the recombinant proteins ⁇ igh3-WT and ⁇ igh3- ⁇ RGD are compared in terms of count (A) and surface area (B) of attached cells.
• Fig. 6a is a histogram showing effects of various compounds on the HCE cell adhesion activities of the recombinant proteins ⁇ igh3-WT and ⁇ igh3- ⁇ RGD.
• Fig. 6b is a histogram showing effects of divalent cations on the HCE cell adhesion activities of the recombinant protein ⁇ igh3-WT.
• Fig. 6c is a histogram showing the inhibition effect of anti-integrin monoclonal antibody on the HCE cell adhesion activity of the recombinant protein ⁇ igh3-WT.
• Fig. 6d is a histogram showing the inhibitory effect of anti-integrin monoclonal antibody on the HCE cell adhesion activities of various proteins.
• Fig. 6e is a histogram showing adhesion specificity of K562 cells for the recombinant protein ⁇ igh3-WT and matrix proteins .
• Fig 7 is a schematic diagram showing recombinant proteins having each of the fas-1 domains of ⁇ ig-h3.
• Fig. 8 is a photograph showing SDS-PAGE results of recombinant proteins containing fas-1 domains of ⁇ ig-h3.
• Fig. 9 is a histogram showing HCE cell adhesion activities of recombinant proteins containing fas-1 domains of ⁇ ig-h3.
• Fig. 10 is a histogram showing the inhibitory effects of anti-integrin antibodies on HCE cell adhesion activities of the recombinant proteins containing fas-1 domains of ⁇ ig- h3.
• Fig. 11 shows parts of amino acid sequences of various matrix proteins containing fas-1 domains.
• Fig. 12 is a schematic diagram showing substitution mutants of the 4 th domain of ⁇ ig-h3.
• Fig. 13 is a photograph showing SDS-PAGE results of recombinant substitution mutants of the 4 th domain of ⁇ ig-h3.
• Fig. 14 is a histogram showing cell adhesion activities of substitution mutants of the 4 th domain of ⁇ ig- h3.
• Fig. 15 is a schematic diagram showing recombinant proteins ⁇ igh3-D-W, ⁇ igh3-D-IV 2X, 3X and 4X containing one, two, three and four copies of the 4 th domain of ⁇ ig-h.3.
• Fig. 16 shows photographs of the recombinant proteins ⁇ igh3-D-TV, ⁇ igh3-D-IV 2X, 3X and 4X run on 10% SDS-PAGE (A) and 8% nondenaturing PAGE (B) , which are purified with the aid of Ni-NTA agarose resin
• Fig. 17 shows optical photographs of wounds to which an ointment base is applied alone (A) and in combination with fibronectin (B) , His- ⁇ -b (C) , and ⁇ ig-h3-D-IV (D) .
• Fig. 18 shows microphotographs of wounds which are in the process of re-epithelialization after being treated with an ointment base alone (A) and in combination with fibronectin (B) , His- ⁇ -b (C) , and ⁇ ig-h3-D-IV (D) .
• Fig. 19 shows optical photographs of wounds which have collagenous fibers formed after being treated with an ointment alone (A) and in combination with fibronectin (B) , His- ⁇ -b (C), and ⁇ ig-h3-D-IV (D) .
• Fig. 20 is a histogram showing HCE cell adhesion activities of the recombinant proteins ⁇ igh3-D-IV, ⁇ igh3-D-IV 2X, 3X and 4X, which contain at least one copy of the 4 th domain of ⁇ ig-h3.
• Fig. 21 shows optical photographs of wounds whose areas are reduced after being treated with a chitosan base alone (A) and in combination with fibronectin (B) , ⁇ ig-h3 3X (C) , and ⁇ ig-h3 4X (D) .
• A chitosan base alone
• B fibronectin
• C ⁇ ig-h3 3X
• D ⁇ ig-h3 4X
• recombinant proteins are prepared on the basis of the 2 nd and 4 th fas-1 domains of ⁇ ig- h3 and used alone or in combination, for cell adhesion and spreading.
• the domains of ⁇ ig-h3 active in cell adhesion and spreading was identified.
• the C-terminal sequence Arg-Gly-Asp (RGD) known as a ligand recognition sequence for several integrins, was examined for its effect on the cell adhesion property of ⁇ ig-h3. The cell attachment activity was measured using the number and surface area of attached cells.
• ⁇ ig-h3 was found to promote cell adhesion and spreading, independent of the RGD motif. Based on this finding, chemical reagents were used to address the specificity of cell adhesion activity of ⁇ ig-h3 and to get further clues about the nature of the cell surface receptor for ⁇ ig-h3.
• the data obtained from the use of chemical reagents suggest that the cell surface receptor for ⁇ ig-h3, which is involved in the cell adhesion activity of ⁇ ig-h3, could be one of the RGD-dependent integrins, which require divalent cations for interaction with ⁇ ig-h3.
• a wound healing method in which the 2 nd and 4 th fas- 1 domains of ⁇ ig-h3 are used, individually or in combination.
• An comparison was made of wound healing effects of mutant ⁇ ig-h3 proteins containing cell adhesion-active domains only and those of wild type ⁇ ig-h3 ( ⁇ ig-h3-WT) containing domains a portion of the domains.
• recombinant proteins containing the cell adhesion-active domains were applied to rats .
• recombinant proteins containing parts of the domains have an advantage in that they can be produced in larger quantities because they are synthesized in water-soluble forms and thus do not undergo denaturation.
• ⁇ ig-h3 cDNA cloned in pBluescript (pBs ⁇ ig-h3) was digested with Ndel and Bglll.
• the DNA fragment was subcloned into the EcoRV-EcoRI site of pET-29b(+) (Novagen Inc.).
• ⁇ igh3-WT was prepared by introducing a 1351 bp Ncol fragment excised from ⁇ ig-h3 cDNA into the Ncol site of this clone.
• ⁇ igh3- ⁇ RGD was derived from ⁇ igh3-WT by cutting out a 3 '-fragment of the ⁇ igh3- T plasmid with AoCI and Notl followed by blunting and self ligation, as shown in Fig. 1.
• E.coli BL 21 DE3 was cultured in LB medium containing 50 ⁇ g/ l kanamycin at 37 °C until the optical density (OD) at 595 nm reached 0.5-0.6.
• the recombinant ⁇ ig-h3 proteins were induced using 1 mM isopropyl- ⁇ -D- (-) - thiogalactopyranoside (IPTG) at 37 °C for 3 hours.
• the pellet thus obtained was resuspended in a lysis buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1 % Triton X- 100, 1 mM PMSF, 0.5 mM DTT) and then sonicated.
• the inclusion bodies were dissolved in a denaturation buffer of 8 M urea containing 20 mM, followed by the purification of the denatured proteins with the aid of Ni-NTA resin (Qiagen) .
• the recombinant proteins were eluted with 200 mM imidazole solution and then dialyzed sequentially from high to low urea in 20 mM Tris-HCl buffer containing 50 mM NaCl. These recombinant proteins were analyzed using SDS-PAGE, as shown in Fig. 2.
• HCE cells used in this assay were cultured in DMEM (EMEM/F-12, Gibco BRL) supplemented with 15% fetal bovine serum, Gentamicin (40 ⁇ g/ml) , insulin (5 ⁇ g/ml), cholera toxin (0.1 ⁇ g/ml) and human epidermal growth factor (hEGF) at 37 °C in 5 % C0 2 .
• DMEM EMEM/F-12, Gibco BRL
• Gentamicin 40 ⁇ g/ml
• insulin 5 ⁇ g/ml
• cholera toxin 0.1 ⁇ g/ml
• hEGF human epidermal growth factor
• the cell adhesion assay was performed as follows . First, the recombinant ⁇ ig-h3 proteins or other extracellular matrix proteins were let to adhere to the bottoms of 96-well microculture plates (Falcon) by incubation at 37 °C for 1 hour and blocked with PBS containing 0.2% BSA.
• the coated extracellular matrix proteins were human plasma vitronectin (Promega) , purified human plasma fibronectin (pFN) , chicken collagen types I and II (Chemicon International Inc.), bovine collagen types IV and VI (Chemicon) , mouse la inin (Chemicon) , and bovine serum albumin (BSA) (Sigma) . Cells were trypsinized and suspended in the culture media at a density of 2xl0 5 cells/ml. 0.1 ml of the cell suspension was added to each well of the plates coated with the recombinant proteins.
• EXPERIMENTAL EXAMPLE 1 Identification of Cell Surface Receptor of ⁇ ig-h3 Involved in Cell Adhesion Activity of ⁇ ig-h3
• HCE cells were preincubated for 30 min in media containing 5 mM EDTA, 100 ⁇ g/ml ⁇ igh3- T, 100 ⁇ g/ml ⁇ igh3- ⁇ RGD, 1 mM RGD, 1 mM RGE or 100 ⁇ g/ml fibronectin, or none of them, and then assayed for cell adhesion as in Example 1.
• Cell adhesion to ⁇ ig-h3 was significantly inhibited by ⁇ ig-h3 itself, RGD peptide and EDTA, and partially inhibited by fibronectin and EGTA, while being not inhibited by RGE peptide.
• Cell adhesion to fibronectin was also significantly inhibited by fibronectin itself, RGD peptide and EDTA, and partially inhibited by ⁇ ig-h3 and EGTA, but not by RGE peptide, as shown in Fig. 6A.
• HBS HEPES-buffered saline
• HCE function-blocking monoclonal antibodies to integrin subunits were examined for their effect on the adhesion of HCE cells to a surface coated with ⁇ ig-h3.
• HCE 3xl0 5 cells/ml
• the preincubated cells were transferred onto plates precoated with ⁇ ig-h3 proteins and then incubated further at 37 °C for 1 hour, followed by the quantitative analysis of ⁇ ig-h3 binding with hexosaminidase substrate.
• the values are expressed as percentages of the number of cells adhering to ⁇ ig-h3 in the absence of monoclonal antibodies.
• Adhesion to the ⁇ ig-h3 coated surface was specifically inhibited by antibody against ⁇ 3 subunit. Because the integrin ⁇ 3 subunit is known to couple with the integrin ⁇ l subunit, anti- ⁇ l antibody significantly blocked cell adhesion to ⁇ ig-h3, as shown in Fig. 6C. Similar results were observed using HT1080 cells.
• As a control experiment for the function-blocking antibodies fibronectin, vitronectin, laminin and type I collagen were employed as substrata. HCE cells were preincubated with function-blocking monoclonal antibodies to integrin subunits and then transferred onto wells coated with 10 ⁇ g/ml fibronectin, vitronectin, type I collagen or laminin. Following incubation, cell counts of adhered cells were analyzed.
• K562 cells known to express ⁇ 5, but not oc3 integrin, were used. K562 cells were inoculated onto plates coated with ⁇ igh3-WT, fibronectin, laminin, or type I collagen and incubated for 1 hour, followed by the hexosaminidase analysis. K562 cells did not adhere to ⁇ ig-h3, but adhered to fibronectin and vitronectin. Taken together, these results suggest integrin ⁇ 3 ⁇ l is a specific receptor for ⁇ ig-h3 in HCE cells, as shown in Fig. 6E.
• E. coli transformants with the expression vectors p ⁇ ig-h3 D-II and p ⁇ ig-h3 D-IV were designated E. coli BL21/His ⁇ -g and E.coli BL21/His ⁇ -e and deposited in the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) with accession Nos. KCTC 0905BP and KCTC 0904BP, respectively, on Dec. 4, 2000.
• the 2 nd and 4 th fas-1 domains were equally active compared to the wild type ⁇ ig-h3 whereas the 1 st fas-1 domain was weak and the 3 rd fas-1 domain was not active at all, as shown in Fig. 9.
• the recombinant protein containing the 4 th fas-1 domain of ⁇ ig-h.3 was mutated by substitution as shown in Fig. 12.
• the substitution mutant of ⁇ ig-h3 D-IV was prepared by PCR and its sequence was confirmed by base sequencing.
• the mutant protein was isolated and purified in the same manner as in Example 1-1 and confirmed on SDS-PAGE, as shown in Fig. 13. Examination was made of the cell attachment activity of the mutated proteins wherein the Pro616, Asp617 and Ile618 of ⁇ igh3 D-IV were, in combination, substituted with Ser, Ala and Ser, respectively.
• the mutant protein in which the three amino acids were mutated named P616S/D617A/I618S ( ⁇ igh3 DIV-sas)
• it also blocked cell adhesion as shown in Fig. 14.
• ⁇ ig-h3 domains active in cell adhesion show the same wound healing function as that of the native ⁇ ig-h3 containing all four fas-1 domains
• various recombinant ⁇ igh3 proteins were prepared as shown in Fig. 15: His- ⁇ -b containing all of 4 fas-1 domains; ⁇ igh3-D- IV containing the 4 th domain alone; and ⁇ igh3-D-IV, 2X, 3X and 4X, each containing at least one 4 th domain.
• the recombinant ⁇ ig-h3 protein His- ⁇ -b was prepared from the recombinant expression vector pET-29 ⁇ anchoring at its EcoRV-EcoRI site an Asp718-BglII fragment which was obtained by deleting a some amino-terminal region from ⁇ ig-h3 cDNA.
• the recombinant proteins His- ⁇ -b and ⁇ igh3-D-IV were expressed and purified in the same manners as in Example 1-1 and 3.
• the recombinant proteins containing at least one 4 th domain such as ⁇ ig3-D-IV, 2X, 3X and 4X were prepared as follows. A DNA fragment encoding to amino acid 498-637 corresponding the 4 th domain was obtained by PCR and the PCR products were blunt-ended by Klenow enzyme. This blunt- ended cDNA fragment was inserted to the EcoRV site of the p ⁇ ig-h3 D-IV, which contained the 4 th domain of ⁇ ig-h3, and the resulting expression vector was named p ⁇ l-h3 D-IV 2X.
• the insert of the p ⁇ ig-h3 D-IV 2X was excised by digestion with EcoRV and Xhol and blunt-ended by treatment with Klenow, followed by inserting the blunt-ended fragment into EcoRV sites of p ⁇ ig-h3 D-IV and p ⁇ ig-h3 D-IV 2X.
• the resulting expression vectors were named p ⁇ ig-h3 D-IV 3X and p ⁇ ig-h3 D- IV 4X. Expression of all recombinant proteins was induced for 3 hours in the presence of 1 mM IPTG and isolated by use of Ni-NTA resin (Qiagen) .
• Isolated recombinant proteins were purified by elution with 20 mM Tris-HCl comprising 50 mM NaCl and 300 mM imidazole.
• ⁇ ig-h3 D-IV 2X, 3X and 4X can be produced in large amounts because they are synthesized as soluble forms, unlike ⁇ ig-h3 recombinant proteins containing all of the four domains, and do not undergo denaturation, as shown in Fig. 16A.
• Electrophoresis using non-denaturing gel revealed that ⁇ ig-h3 D-IV did not form polymers while 2X partially formed polymers and 3X and 4X each readily formed polymers , as shown in Fig. 16B.
• E. coli BL21/His ⁇ -e4X which harbors the expression vector p ⁇ ig-h3 D-IV 4X containing four 4 th domains of ⁇ ig-h3, was deposited in the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) with accession No. KCTC 0906BP on Dec. 4, 2000.
• Fibronectin serving as a positive control, was purified from citrated rat plasma by affinity chromatography using gelatin-sepharose 4B.
• the plasma was filtered at room temperature through non-substituted sepharose 4B and the eluate was loaded onto gelatin sepharose 4B equilibrated with 0.05 M Tris-Cl containing 0.05 M EACA ( ⁇ -amino caproic acid), 0.02 M sodium citrate and 0.02 % sodium azide. After being eluted, most plasma proteins were washed with a buffer containing 1 M sodium chloride.
• absorbed fibronectin was eluted with 3M uric acid isotonic buffer which was subsequently dialyzed for about 48 hours against PBS, pH 7.2, to purify fibronectin. Its concentration was determined by UV absorbance at 280 nm and freeze-dried before being stored at -20 °C.
• 1-A, 1-B, 1-C and 1-D according to the ointment applied thereto .
• 1-A coated at a dose of 1 gm per day with an ointment base combined with no materials.
• 1-B coated at a dose of 1 gm per day with an ointment in which fibronectin was combined at a concentration of 100 ⁇ g/ml with a base.
• 1-C coated at a dose of 1 g per day with an ointment in which His ⁇ -b protein was combined at a concentration of
• 1-D coated at a dose of 1 g per day with an ointment in which ⁇ igh3-D-IV protein was combined at a concentration of 100 ⁇ g/ml with a base.
• Ointments for test groups 1-A, 1-B, 1-C and I-D were applied at an amount of about 1 g to the wounds which were then covered with a synthetic dressing (Tegaderm ® 3M) and lightly bandaged. Application of ointments was performed once every day.
• each of the ointments contained, per 1 g, spermaceti 38 mg, stearyl alcohol 116 mg, polyethylglycol 38 mg, cone, glycerin 192 mg, ethanol 23 mg, lauryl sodium sulfate mg, ethyl paraoxybenzoate 0.87 mg, butyl paraoxybenzoate 0.12 mg, and purified water.
• collagenous fiber formation As for collagenous fiber formation, it was graded as 1+ for insignificant formation of collagenous fibers as observed with trichrome dye, 2+ for scatteringly spaced collagenous fibers, and 3+ for dense collagenous fibers.
• the recombinant proteins ⁇ ig-h3 D-IV 2X, 3X and 4X were all found to effectively induce the adhesion of HCE cells.
• the following experiments were conducted.
• test group 2 four circular dermal whole layer wounds were made on the back of each rat and coated with chitosan bases combined with materials of interest: 2-A: wound coated with chitosan base only 2-B: wound coated with chitosan base in combination with 500 ⁇ g/ml of fibronectin
• the composites based on chitosan were prepared as follows. 1 g of water soluble chitosan (poly (1-4) 2-amino- 2-deoxy- ⁇ -D-glucan) with a molecular weight of 600,000 Da was dissolved in 100 ml of sterile distilled water and the resulting 1% solution was dispensed in aliquot of 2 ml to to 12-well plate (Corning, USA), followed by the addition of 100 ⁇ g of gentamycin per well.
• Fibronectin, ⁇ igh3-D-IV 3X, and ⁇ igh3-D-IV 4X were individually added to a concentration of 500 ⁇ g/ml and frozen at -70 °C, followed by freeze-drying in a freeze drier (Ilshin) for 12 hours to give disc-shaped composites .
• a high wound healing effect was obtained from the composite containing the recombinant protein ⁇ ig-h3 D-IV 3X or 4X.
• the recombinant proteins of the present invention which contain the 2 nd and 4 th domains of ⁇ ig-h3, alone or incombination, or in multiplicate are effective for cell adhesion and wound healing and ultimately can be utilized in developing cell culture and wound healing agents.
• recombinant proteins containing at least one of the 2 nd and 4 th domains of ⁇ ig-h3 in which one aspartic acid and one isoleucine residue, known to be essential for association with integrin, are highly conserved. Also, the recombinant proteins themselves are useful for cell adhesion and wound healing, making a contribution to the development of- cell culture methods and wound healing agents.
• microorganism identified under 1 above wa$ accompanied by:

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