US20060073566A1 - Active modified hedgehog proteins - Google Patents

Active modified hedgehog proteins Download PDF

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US20060073566A1
US20060073566A1 US11/273,989 US27398905A US2006073566A1 US 20060073566 A1 US20060073566 A1 US 20060073566A1 US 27398905 A US27398905 A US 27398905A US 2006073566 A1 US2006073566 A1 US 2006073566A1
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protein
activity
hedgehog
mutant
hedgehog protein
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Kurt Lang
Ulrike Leser
Tilmann Seytter
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Curis Inc
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Curis Inc
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/04Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00Uses of viruses
    • C12N2799/02Uses of viruses as vector
    • C12N2799/021Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the invention concerns an active form of a hedgehog protein, a process for its recombinant production and its therapeutic use.
  • Hedgehog (hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349).
  • hh family is composed of at least three members i.e. sonic, indian and desert hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51).
  • Hynes et al. compare the activity of hh in the supernatant of transformed human embryonic kidney 293 cells (eukaryotic hh) with hh produced from E. coli and isolated from the cytoplasm and find a four-fold higher activity of hh from the supernatants of the kidney cell line.
  • Nakamura et al. compare the activity of shh in the supernatant of transformed chicken embryo fibroblasts with an shh fusion protein isolated from E. coli which still has an N-terminal polyhistidine part.
  • the shh in the supernatant of the fibroblasts has a seven-fold higher activity than the purified E: coli protein with regard to stimulation of alkaline phosphatase (AP) in C3H10T 1 ⁇ 2 cells.
  • Molecules such as bone morphogenetic proteins (BMPs) have been discussed as the reason for the increased activity which are only present in the supernatant of eukaryotic cells and cause the stronger induction of AP.
  • the object of the invention is to produce hh proteins (polypeptides) which have a considerably improved activity compared to the known forms.
  • the highly active hh proteins are particularly useful for inducing or stimulating chondrocytes and osteocytes as well as for treating neurodegenerative diseases.
  • the highly active hh proteins of the present invention are useful therapeutic agents for treating bone disorders such as, for example, osteoporosis and bone fractures.
  • a method for obtaining isolated highly active post-translationally processed hedgehog protein mutants (hh mutant) which are esterified with a fatty acid having from 14 to 20 carbon atoms (i.e., C 14 -C 20 ) at the N-terminal domain of the protein.
  • the highly active hh proteins of the present invention are produced by expression of a gene which encodes a hedgehog protein using a baculovirus expression system in a fermentation medium capable of producing the esterified hedgehog protein wherein the fermentation period is up to 30 hours, preferably from about 24 to about 27 hours.
  • Isolation of the hh mutant from the fermentation supernatent can be obtained by conventional protein isolation techniques such as through binding of the hh protein to heparin-Sepharose and hydroxylapatite. Purification of the fermentation supernatant is preferably performed in the presence of a protease inhibitor and a non-ionic detergent
  • AP alkaline phosphatase
  • FIG. 2 is an elution diagram of the purification of the fermentation supernatant with heparin-Sepharose.
  • FIG. 3 is an elution diagram of the purification of the dialyzed eluate of the heparin-Sepharose with hydroxylapatite.
  • FIG. 4 is an elution diagram of the purification of the dialyzed active fractions of the hydroxylapatite column with a 1 ml HiTrap heparin column.
  • FIG. 5 shows the alkaline phosphatase inducing activity of the fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 6 shows coomassie staining of SDS-PAGE with alkylated fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 7 is a Western blot with an antibody against the N-terminus of shh of the SDS-PAGE with alkylated samples of the fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 8 is a Western blot with an antibody against the N-terminus of shh of the SDS-PAGE with reducing samples of the fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 9 shows the influence of suramin on the activity of the hh mutant: No suramin (B), suramin ad 0.1 mg/ml only added after dialysis against PBS+0.05% Tween®80 or (C) suramin ad 0.1 mg/ml added before dialysis and dialysed against PBS+0.05% Tween®80 containing additionally 0.1 mg/ml suramin (D) were added to aliquots of an active fraction after hydroxylapatite chromatography.
  • the AP activity in the absence of hh is shown by (A).
  • FIG. 10 shows the influence of Tween®20 and Tween®80 on the activity of the hh mutant: Aliquots of a pool of AP active fractions after SP Sepharose chromatography in 50 mM NaPi, 0.9 M NaCl, 1 mM EDTA pH 7.3 were admixed with the stated concentrations of Tween and dialysed against PBS containing the respective concentration of Tween. The samples were sterile filtered through 0.2 ⁇ m filters before being used in the C3H10T1 ⁇ 2 test.
  • FIG. 11 shows the influence of trypsin and chymotrypsin on the activity of the hh mutant: AP active fractions after a step elution of heparin-Sepharose were adjusted to a protein concentration of 0.46 mg/ml in 10 mM Na phosphate, 0.05% Tween®80 and admixed with trypsin or chymotrypsin at a protease/protein ratio (w/w) of 1:100 (A), 1:500 (B), 1:2500 (C) and 1:10000 (D). The samples were incubated for 11 h at RT.
  • FIG. 12 is an elution diagram of the purification of dialysed active fractions of the hydroxylapatite column with a 1.7 ml Poros HS/M column.
  • the UV-detected elution diagram is shown as a line and the activities of the fractions in the cell test are shown as bars.
  • FIGS. 13A and 13B are elution diagrams of the separation of the active fractions of the Poros HS/M column by means of RP-HPLC.A.: Elution diagram of 10 to 75 min. detected at 220 nm. B: Elution diagram of 15 to 40 min. detected at 280 nm.
  • FIG. 14 is an elution diagram of the purification of the dialysed active fractions of the hydroxylapatite column with a 6 ml Poros Q column.
  • the UV signal of the elution is shown as a line, the activity in the cell test as bars.
  • FIG. 15 shows the stability of the alkaline phosphatase inducing activity towards dithiothreitol.
  • FIG. 16 shows the stability of the alkaline phosphatase inducing activity towards hydroxylamine.
  • FIG. 17 shows MALDI mass spectra of the active modified hh derivative after RP-HPLC.
  • A, B Spectra obtained with different laser energies and from different spots.
  • the present invention provides isolated highly active hedgehog proteins having a molecular weight of from about 19 to about 26 kD which are esterified with a fatty acid having from 14 to 20 carbon atoms at the N-terminal domain of the protein. Esterification of the hedgehog protein with a C 16 fatty acid is preferred.
  • the highly active hh proteins are particularly useful for inducing or stimulating chondrocytes and osteocytes as well as for treating neurodegenerative diseases.
  • the highly active hh proteins of the present invention are useful therapeutic agents for treating bone disorders such as, for example, osteoporosis and bone fractures.
  • the method of the present invention for producing the highly active hedgehog proteins esterified with a fatty acid having from 14 to 20 carbon atoms comprises (a) providing an insect cell containing a baculovirus vector having a gene inserted therein capable of expressing a hedgehog protein, and a fermentation medium such that the insect cell and medium are capable upon fermentation of producing a fatty acid having from 14 to 20 carbon atoms; (b) fermenting the insect cell in the medium for a period of about 30 hours or less to produce the fatty acid and express the hedgehog protein esterified with the fatty acid; and (c) isolating the esterified hedgehog protein from the protein produced during the fermentation.
  • Any fermentation conditions which allow the esterification of the hedgehog protein with a fatty acid having from 14 to 20 carbon atoms can be utilized.
  • the fermentation period is from about 24 to about 27 hours.
  • the fermentation is carried out at from about 10 to 35° C. and pH of about 4 to 8. Fermentation at room temperature and neutral pH is preferred.
  • Any conventional medium for fermentation of the baculovirus expression system that allows the esterification of the hh protein can be utilized.
  • a preferred fermentation medium is Excell 400 medium (JHR, Inc.).
  • Isolation of the esterified hh protein can be obtained by conventional techniques for isolation of proteins such as through binding of the hh protein to heparin-Sepharose and hydroxylapatite.
  • the hedgehog proteins of the present invention can be further purified by techniques such as anion or cation exchange chromatography and reverse-phase HPLC. Purification of the cell supernatant is preferably performed in the presence of a protease inhibitor and a non-ionic detergent.
  • Hedgehog proteins are known and any hedgehog protein can be modified in accordance with this invention to esterify the protein at the N-terminal domain with a fatty acid having from 14 to 20 carbon atoms and in so doing provide the highly active hh proteins of the present invention.
  • a baculovirus expression system is understood as an expression system composed of a baculovirus vector and an insect cell as the host cell.
  • Such expression systems are known to a person skilled in the art and are for example described by Bumcrot (1995) for hh proteins.
  • Preferred insect cells for use in obtaining the esterified hedgehog proteins of the present invention are High five cells.
  • a hedgehog protein is understood by the invention as a secreted signal protein which is responsible for the formation of numerous structures in embryogenesis. Sonic, indian or desert hh proteins are particularly preferably used (M. Fietz et al. (1994). A hh protein with a sequence as described in the EMBL database under the No. L38518 is preferably used. Proteins of the hedgehog family exhibit a pronounced homology in their amino acid sequence which is why it is also preferable to express those nucleic acids which code for hedgehog proteins which are 80% or more homologous with the above-mentioned sequence of sonic hedgehog protein.
  • the human sonic hedgehog precursor protein is composed of the amino acids 1-462 of the sequence described in the EMBL database under No. L38518.
  • the amino acids 1-23 represent the signal peptide
  • the amino acids 24-197 represent mature signal domain
  • the amino acids 32-197 represent the signal domain shortened by 8 amino acids
  • the amino acids 198-462 represent the auto-processing domain after autoproteolytic cleavage.
  • amino acids 24-197 of the human sonic hedgehog protein described in EMBL database sequence No. L38518 represent the N terminal domain of this sequence.
  • the first 8 amino acids of the hedgehog proteins of the present invention are the first 8 amino acids of the processed hedgehog protein, for example, the amino acids Cys24-Gly31 of the sequence described in the EMBL database under No. L38518 for sonic hedgehog protein. Since the modified group can be cleaved with the first N-terminal amino acids of hedgehog protein and the activity is greatly reduced by incubation with hydroxylamine or DTE, the binding of the group is localized on these amino acids preferably in the form of a thioester, preferably as palmitic acid thioester, on the cysteine which is present in the first eight amino acids of the hedgehog protein.
  • a highly active form of the protein (activity increased by at least 10-fold, preferably at least 100-fold compared to recombinant shh from the cytoplasm of E. coli ) accumulates in the initial period of the fermentation.
  • the amount of highly active hh mutant according to the invention is only about 0.2-5% of the total protein in the supernatant of the cells after expression in the baculovirus expression system.
  • This mutant of the polypeptide according to the invention can be in particular isolated when the fermentation is terminated at the latest after about. 30 hours or less, preferably after about 24-27 hours.
  • hedgehog proteins that although the amount of hedgehog protein in the fermentation supernatant greatly increases in the period between 33 and 72 hours, mainly hh protein with an activity that is known from the prior art is formed in this period. In contrast the amount of such a hh protein is considerably less (at least 3-5-fold) when the fermentation period is reduced to below ca. 30 h. which allows the identification and isolation of the highly active hh protein mutant according to the invention.
  • the molecular weight of the mutant according to the invention is 19,796.7 ⁇ 2 D when analysed by means of MALDI mass spectroscopy and is increased by 236.7 ⁇ 2 D compared to unmodified hh protein (cytoplasmically expressed hh protein in E. coli ), corresponding to the molecular weight of a palmitic acid thioester.
  • the hydrophobic modification increases the mobility in SDS-PAGE by increased SDS binding so that apparently a lower molecular weight is seen under alkylating conditions (derivatized hh protein) than under strongly reducing conditions (hh protein without derivatization).
  • the accuracy of the molecular weight determination in SDS-PAGE is also about ⁇ 1-2 D.
  • the hh mutant according to the invention After purification with heparin-Sepharose, hydroxyl-apatite and porous HSM ion exchanger chromatography, the hh mutant according to the invention has an activity measured via the induction of alkaline phosphatase in a cell test (activity in the AP cell test) which is increased by at least 50-fold, preferably by at least 100-fold and particularly preferably by at least 10 3 to 10 6 -fold compared to soluble hh protein expressed in the cytoplasm of E. coli .
  • Such an active hh mutant is not modified by cholesterol like the N-terminal hh fragment described by J. A. Porter since only the N-terminal and not also the C-terminal autoprocessing domain was expressed.
  • the hh mutant according to the invention is present in a biologically active three-dimensional structure. Consequently the invention for the first time enables the isolation of highly active hedgehog protein and provides a general, reproducible method for the production and characterization of highly active hedge
  • the invention concerns hedgehog proteins with an at least 100-fold, preferably at least 10 3 to 10 6 -fold increased activity compared to the corresponding hedgehog protein which was isolated from the cytoplasm of E. coli were the activity is determined by the induction of alkaline phosphatase in the cell test.
  • a corresponding hedgehog protein which is produced in the cytoplasm of E. coli is understood according to the invention as a hedgehog protein which is isolated in a soluble form in the cytoplasm after expression in E. coli .
  • a vector is used as the expression vector which contains a nucleic acid to be expressed which codes for a hedgehog protein of the same amino acid sequence as the nucleic acid to be expressed of the expression vector which is used for expression in the baculovirus expression system.
  • the hh protein that is formed in this process is not post-translationally modified (no derivatization with cholesterol etc.).
  • the hh mutant according to the invention is very sensitive towards proteases which is why it is preferable to add protease inhibitors such as for example aprotinin, EDTA (up to 1 mmol/l), PMSF or pepstatin or a mixture thereof to the supernatant of the fermentation.
  • protease inhibitors such as for example aprotinin, EDTA (up to 1 mmol/l), PMSF or pepstatin or a mixture thereof to the supernatant of the fermentation.
  • non-ionic detergents such as polysorbate (e.g. Tween®20, Tween®80, Triton®X100) during the purification, preferably before or after the first crude purification over heparin-Sepharose. Since this also stabilizes the hh proteins according to the invention.
  • polysorbate e.g. Tween®20, Tween®80, Triton®X100
  • a chromatography on heparin-Sepharose it is expedient to carry out a chromatography on heparin-Sepharose. It is preferable to carry out this chromatography as a step elution i.e. preferably to elute at a concentration of at least 0.7 mol/l NaCl (preferably 1.2 mol/l) after washing with 250 mmol/l NaCl.
  • a hydroxylapatite chromatography it is particularly preferable to carry out a hydroxylapatite chromatography to purify the hh mutant according to the invention. This achieves a good concentration of the activity with relatively low losses ( ⁇ 50%).
  • Further suitable chromatographic steps are for example a heparin-Sepharose chromatography (Miao et al., J. Neurosci. 17 (1997) 5891-5899) which is, however, preferably carried out in the presence of non-ionic detergents.
  • the pH value of the buffer is at least pH 5, in particular pH 6.5-7.5 and the ionic strength of the buffer corresponds to a solution of 1-20 mM sodium phosphate and 10-100 mM NaCl in particular 50 mM NaCl and the dialysis is carried out at a low concentration of total protein (1 mg/ml or less, preferably 0.5 mg/ml or less).
  • suramin during the purification or at least before determining the activity of the protein.
  • the activity can also be stabilized by adding serum albumin (at least 50 ⁇ g/ml to 5 mg/ml) to the sample before dilution in the cell test. This also stabilizes the activity.
  • serum albumin at least 50 ⁇ g/ml to 5 mg/ml
  • suramin inhibits the activity of growth factors (Middaugh et al., Biochem. 31 (1992) 9016-9024). Surprisingly it was found that the activity of hh proteins is increased by adding suramin.
  • the hh mutant according to the invention can be used to produce a pharmaceutical composition which is also a subject matter of the invention.
  • This pharmaceutical composition contains a pharmacologically effective dose of the protein according to the invention and can be administered systemically as well as locally.
  • the proteins according to the invention in combination wish other proteins of the hedgehog family or bone growth factors such as bone morphogenetic proteins (BMPs), (Wozney et al., Cell. Mol. Biol. of Bone, Bone Morphogenetic Proteins and their Gene Expression, 131-167, Academic Press Inc. 1993) or parathyroid hormones (Karablis et al., Genes and Development 8 (1994) 277-289).
  • BMPs bone morphogenetic proteins
  • the protein according to the invention can be used advantageously to induce or stimulate chondrocytes and osteocytes in an osteoinductive pharmaceutical composition as well as to treat neurodegenerative diseases.
  • Osteoinductive pharmaceutical compositions are for example known from the U.S. Pat. No. 5,364,839, WO 97/35607, WO 95/16035.
  • the protein according to the invention When the protein according to the invention is administered locally it is preferable to use it in combination with a suitable matrix as a carrier and/or with a sequestering agent.
  • a suitable matrix is suitable for slowly releasing the protein in vivo in an active form in particular in the vicinity of bone and cartilage.
  • the sequestering agent is a substance which facilitates administration for example by injection and/or prevents or at least delays migration of the protein according to the invention from the site of administration.
  • a biocompatible degradable material for example based on collagen or other polymers based on polylactic acid, polyglycolic acid or co-polymers of lactic acid and glycolic acid are particularly suitable as a matrix material.
  • Such polymer matrices are described for example in WO 93/00050.
  • Sequestering agents are for example cellulose and cellulose-like materials and for example alkyl cellulose, carboxymethyl cellulose, hyaluronic acid, sodium alginate, polyethylene glycol and polyvinyl alcohol of which hyaluronic acid is particularly preferred especially in a pharmaceutical composition even without carrier matrix.
  • auxiliary substances such as mannitol, sucrose, lactose, glucose or glycine and antioxidants such as EDTA, vitamin C, citrate and detergents, preferably non-ionic detergents like polysorbates and polyoxyethylenes.
  • auxiliary substances such as mannitol, sucrose, lactose, glucose or glycine and antioxidants such as EDTA, vitamin C, citrate and detergents, preferably non-ionic detergents like polysorbates and polyoxyethylenes.
  • a pharmaceutical composition of the hedgehog protein according to the invention together with suramin and/or serum albumin is preferred and this is advantageously used.
  • the N-terminal domain of human shh with the amino acids 24-197 was expressed with the N-terminal signal peptide 1-23 as described by Miao (J. Neurosci. (1997) 17, 5891-5899) and Bumcrot et al., (Mol. Cell. Biol. (1995) 15, 2294-2303) for the rat protein by means of recombinant baculovirus in High Five cells (Invitrogen, Leek, N L, Order No. E 855-02) using Excell 400 medium (JHR, Inc.) in which sufficient virus was used to infect each cell on average with one virus (multiplicity of infection (m.o.i.):1).
  • the fermenter contents were clarified after 26 or 72 h by centrifugation at 1000 g and filtration and the supernatant or the permeate was stored at -80° C. until further use. Fermentation samples were analysed for their content of alkaline phosphatase inducing activity [Nakamura et al. (1997), Kinto et al. (1997) FEBS Lett. 404, 319-323] and for their content of shh protein by means of RP-HPLC (Vydac C18, gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid, TFA) or SDS-PAGE.
  • RP-HPLC Vydac C18, gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid, TFA
  • the fermentation was terminated after 24-32 h (preferably after 24-27 h) fermentation time and the supernatant was clarified.
  • the dialysate was applied to a hydroxyapatite column (volume 10 ml; Makro Prep; 40 ⁇ m, type I; BIO-Rad) equilibrated with buffer B. It was eluted with a gradient of 10 to 300 mM NaP in buffer B (2 ⁇ 200 ml).
  • the active fractions were pooled and dialysed against buffer B at 4° C. and applied to a 1 ml HiTrap heparin column (Pharmacia Biotech) which had been equilibrated with 20 mM potassium phosphate, 0.05 Tween®80, pH 7.2. It was eluted by a gradient of 20-1400 mM KCl in 20 mM potassium phosphate, 0.05% Tween®80, 50 mM NaCl, pH 7.2. Active fractions were identified by the stimulation of alkaline phosphatase in C3H10T1 ⁇ 2 cells, and alkylated and reduced samples were analysed by means of SDS-PAGE and Western blot with an antibody against the N-terminus of shh.
  • Active fractions of the Poros®-HS/M chromatography were further purified by RP-HPLC. For this 3.2 ml of a highly active fraction was applied to a 2.1 ⁇ 150 mm butyl column (VydacTM 214TP5215) which had been equilibrated in 20% acetonitrile, 0.1% trifluoroacetic acid (TFA). It was eluted at 25° C. in a gradient of 20-90% acetonitrile in 0.1% TFA and analysed by detecting the absorbance at 220 nm and 280 nm.
  • VydacTM 214TP5215 2.1 ⁇ 150 mm butyl column
  • TFA trifluoroacetic acid
  • the concentrated highly active hh derivative did not elute until a somewhat higher concentration of acetonitrile (ca. 41.2%). This species was collected and its mass was determined by MALDI mass spectrometry.
  • the RP eluate described above (total volume 200 ⁇ l) was admixed with 5 ⁇ l 25 mM sinapinic acid in 30% (w/w) acetonitrile/70% water/0.1% trifluoro-acetic acid, evaporated to dryness in a Speedvac concentrator and dissolved in 5 ⁇ l 30% (v/v) acetonitrile/70% water/0.1% trifluoroacetic acid.
  • solution A 1 ⁇ l of the solution obtained in this manner was mixed with 1 ⁇ l 25 mM sinapinic acid in 30% (v/v) acetonitrile/70% water/0.1% trifluoroacetic acid, applied to the target, dried in laboratory air and after drying it was measured in a Bruker REFLEX MALDI mass spectrometer with a so-called delayed extraction source. Only one molecular species was detected in the mass spectrum obtained in this manner apart from alkali and matrix adducts.
  • the difference between the molecular mass determined for the active hedgehog molecule and the molecular mass of the unmodified hedgehog molecule is 236.7 ⁇ 2 D.
  • the known naturally occurring covalent protein modifications only esterification with C16 fatty acids (palimitic acid: mass increase by 238.4 D, monounsaturated palmitic acids: mass increase by 236.4 D) leads to an increase of the molecular mass which is compatible with the found increase in mass (Turner and Smith, Molecular Biotechnol. 8 (1997) 233-249).
  • Active hh protein eluted at a salt content of 90-175 mM NaCl. These active fractions can be further purified and characterized by means of Poros®-HS/M or RP-HPLC or SDS-PAGE and Western blot as described above.
  • the step eluate of a heparin-Sepharose column containing buffer A which additonally contained 1.2 M NaCl (see example 3) was diluted with 1 volume 0.05% Tween®80 and 1/10 vol. 1 M Tris/HCl, pH 8 was added for the samples at pH 8.
  • DTT was added to the samples at final concentrations of 0 mM, 1 mM, 10 mM and 50 mM.
  • each of the samples was admixed with 1/10 volumes (50 ⁇ l) 10 mg/ml BSA and dialysed against PBS. Before use in the C3H10T1 ⁇ 2 cell test, a final concentration of 0.1 mg/ml suramin was added to each sample in order to stabilize the remaining activity.
  • the step eluate of a heparin-Sepharose column with buffer A which additionally contained 1.2 M NaCl (see example 3) was adjusted with NaOH to pH 8.0 or with HCl to pH 5.5. Aliquots of these samples were each admixed with 0 mM, 66 mM, 250 mM or 1 M of NH 2 OH with the corresponding pH value and incubated for 14 h at RT. The samples were subsequently dialysed against 20 mM NaP, 250 mM NaCl, 0.05% Tween80, pH 7.4 and admixed with 1 mg/ml (final concentration) BSA and 0.1 mg/ml (final concentration) suramin before analysis in the cell test.
  • C3H10T1 ⁇ 2 (ATCC CCl-226) were sown in each well of a 96-well microtitre plate.
  • the cells were in 100 ⁇ l DMEM, 2 mM glutamine, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin and 10% foetal calf serum, FCS.
  • active substances to be examined were added at the appropriate concentrations in a volume of 100 ⁇ l after 20-500-fold predilution in culture medium. The test was stopped after 5 days. For this purpose the supernatants were discarded and the cells were washed once with PBS.
  • the cells were lysed in 50 ⁇ l 0.1% Triton®X-100 and frozen at ⁇ 20° C. After thawing 25 ⁇ l was used for the protein determination and 25 ⁇ l for the determination of the activity of alkaline phosphatase.
  • reaction buffer 100 ⁇ l reaction buffer (Sigma 221) was added to the preparation.
  • a substrate capsule (Sigma 104-40) was dissolved in 10 ml redistilled H 2 O and then 100 ⁇ l was added to the test mixture by pipette. The OD was measured at 405 nm after the yellow coloration.
  • alkaline phosphatase converts p-nitrophenyl phosphate into p-nitrophenol.
  • the ODs were each converted into nmol or ⁇ g by means of standard curves.
  • the evaluation was according to the formula:
  • hh protein was used at a defined concentration in the C3H10T1 ⁇ 2 cell test.
  • the protein determination was carried out for the unmodified hh protein from E. coli by means of its UV absorbance at 280 nm (Mach, H., et al., Anal. Biochem. 200 (1992) 74-80).
  • the concentration of the hh derivative purified from the baculovirus fermentation supernatant was determined by means of RP-HPLC.
  • the concentration in the stock solution was determined by integrating the area under the absorbance curve of the hh peak in the RP-HPLC where the detection was carried out at 220 and 280 nm and a calibration curve was established by an analogous chromatography using stock solutions of unmodified shh of known concentration.
  • the relative activity of the isolated shh proteins was determined in the C3H10T1 ⁇ 2 cell test by determining the stimulation of the expression of alkaline phosphatase in these cells (example 5; cell test) compared to the baculovirus fermentation supernatant (BVCM after 24 hour fermentation) in a 1:40 dilution where the individual values were corrected for the base line activity of the cells in the absence of added shh. This determination of the relative activity is preferred since the stimulatability of the cells is influenced by the medium used and the preculture of the cells.
  • Table 2 shows the relative activities of purified, unmodified shh from E. coli and of the shh derivative purified from the supernatant of the baculovirus fermentation supernatant, which had been purified by chromatography on heparin-Sepharose, hydroxylapatite and Poros HS/M.
  • hh protein purified from E. coli must be present at a concentration of ca. 240 ⁇ g/ml but the hh protein derivative only needs to be present at a concentration of ca. 2.3 ng/ml in the cell test in order to reach the same alkaline phosphatase activity as the BVCM at a 1:40 dilution.
  • the hh protein derivative has a ca. 10 4 -10 5 -fold higher specific activity than the unmodified hh protein.
  • modified and unmodified shh protein was examined in an animal model established for bone growth factors (Mackie & Trechsel, Bone 11 (1990) 296; Kling, L., et al., J. Bone Min. Res. 11 (Suppl. 1) (1996) 153). Seven week old female BALB/c mice were subcutaneously injected daily over a period of 15 days with 1, 10 or 50 ⁇ g shh in a volume of 50 ⁇ l into the cranial calotte. 14 days after completion of the treatment, the calottes were removed and purified of surrounding connective tissue. Subsequently the weights of the standardized explants and the X-ray density were analysed. Modified shh exhibited a higher osteoanabolic effect than unmodified shh.
  • the AP active fractions were admixed with trypsin or chymotrypsin at different protease/protein ratios. After incubation of the samples for 20 h at 25° C. the digestion was stopped by addition of aprotinin and the samples were analysed by means of SDS-PAGE and in the C3H10T1 ⁇ 2 test (see FIG. 11 ).
  • the hedgehog protein is degraded to a shortened form which migrate at 20 kDa in the SDS-PAGE.
  • amino-terminal sequencing and MALDI mass spectrometry it could be shown that the mature hh protein is degraded by the chymotrypsin or trypsin treatment at a protease/protein ratio (w/w) of 1:100 to fragments in which the first 7 or 14 amino-terminal amino acids are absent.
  • the alkaline phosphatase inducing activity is also reduced when the hh protein is shortened.

Abstract

The present invention provides isolated highly active hedgehog proteins esterified with a fatty acid having from 14 to 20 carbon atoms at the N-terminal domain of the protein. The highly active hh proteins are particularly useful therapeutic agents for treating bone disorders and neurodegenerative diseases. Methods for obtaining the highly active modified hedgehog proteins are also provided.

Description

    FIELD OF THE INVENTION
  • The invention concerns an active form of a hedgehog protein, a process for its recombinant production and its therapeutic use.
  • BACKGROUND OF THE INVENTION
  • Hedgehog (hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349). During its biosynthesis a 20 kD N-terminal domain and a 25 kD C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. The N-terminal fragment is modified in its natural form with cholesterol at its C-terminus (J. A. Porter et al., Science 274 (1996) 255-259). In higher life-forms the hh family is composed of at least three members i.e. sonic, indian and desert hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of hedgehog proteins that were produced recombinantly were observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997) 465-469.
  • Hynes et al. compare the activity of hh in the supernatant of transformed human embryonic kidney 293 cells (eukaryotic hh) with hh produced from E. coli and isolated from the cytoplasm and find a four-fold higher activity of hh from the supernatants of the kidney cell line. A potential additional accessory factor which is only expressed in eukaryotic cells, a post-translational modification, a different N-terminus since the hh isolated from E. coli contains 50% of a hh which carries two additional N-terminal amino acids (Gly-Ser) or is shortened by 5-6 amino acids, or a higher state of aggregation (e.g. by binding to nickel agarose beads) have been discussed to be the reason for this increased activity.
  • Nakamura et al. compare the activity of shh in the supernatant of transformed chicken embryo fibroblasts with an shh fusion protein isolated from E. coli which still has an N-terminal polyhistidine part. The shh in the supernatant of the fibroblasts has a seven-fold higher activity than the purified E: coli protein with regard to stimulation of alkaline phosphatase (AP) in C3H10T ½ cells. Molecules such as bone morphogenetic proteins (BMPs) have been discussed as the reason for the increased activity which are only present in the supernatant of eukaryotic cells and cause the stronger induction of AP.
  • Kinto et al., FEBS Letters, 404 (1997) 319-323 describe that fibroblasts which secrete hh induce ectopic bone formation in an i.m. implantation on collagen.
  • SUMMARY OF THE INVENTION
  • The object of the invention is to produce hh proteins (polypeptides) which have a considerably improved activity compared to the known forms. In accordance with the present invention, the highly active hh proteins are particularly useful for inducing or stimulating chondrocytes and osteocytes as well as for treating neurodegenerative diseases. Thus, the highly active hh proteins of the present invention are useful therapeutic agents for treating bone disorders such as, for example, osteoporosis and bone fractures.
  • In accordance with the present invention, a method is provided for obtaining isolated highly active post-translationally processed hedgehog protein mutants (hh mutant) which are esterified with a fatty acid having from 14 to 20 carbon atoms (i.e., C14-C20) at the N-terminal domain of the protein. The highly active hh proteins of the present invention are produced by expression of a gene which encodes a hedgehog protein using a baculovirus expression system in a fermentation medium capable of producing the esterified hedgehog protein wherein the fermentation period is up to 30 hours, preferably from about 24 to about 27 hours.
  • Isolation of the hh mutant from the fermentation supernatent can be obtained by conventional protein isolation techniques such as through binding of the hh protein to heparin-Sepharose and hydroxylapatite. Purification of the fermentation supernatant is preferably performed in the presence of a protease inhibitor and a non-ionic detergent
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a plot showing the kinetics of the secretion of alkaline phosphatase (AP) inducing activity (bars) and shh protein (dots and line) by High Five cells after infection with baculovirus (t=0).
  • FIG. 2 is an elution diagram of the purification of the fermentation supernatant with heparin-Sepharose.
  • FIG. 3 is an elution diagram of the purification of the dialyzed eluate of the heparin-Sepharose with hydroxylapatite.
  • FIG. 4 is an elution diagram of the purification of the dialyzed active fractions of the hydroxylapatite column with a 1 ml HiTrap heparin column.
  • FIG. 5 shows the alkaline phosphatase inducing activity of the fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 6 shows coomassie staining of SDS-PAGE with alkylated fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 7 is a Western blot with an antibody against the N-terminus of shh of the SDS-PAGE with alkylated samples of the fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 8 is a Western blot with an antibody against the N-terminus of shh of the SDS-PAGE with reducing samples of the fractions of the 1 ml High Trap heparin chromatography.
  • FIG. 9 shows the influence of suramin on the activity of the hh mutant: No suramin (B), suramin ad 0.1 mg/ml only added after dialysis against PBS+0.05% Tween®80 or (C) suramin ad 0.1 mg/ml added before dialysis and dialysed against PBS+0.05% Tween®80 containing additionally 0.1 mg/ml suramin (D) were added to aliquots of an active fraction after hydroxylapatite chromatography. The AP activity in the absence of hh is shown by (A).
  • FIG. 10 shows the influence of Tween®20 and Tween®80 on the activity of the hh mutant: Aliquots of a pool of AP active fractions after SP Sepharose chromatography in 50 mM NaPi, 0.9 M NaCl, 1 mM EDTA pH 7.3 were admixed with the stated concentrations of Tween and dialysed against PBS containing the respective concentration of Tween. The samples were sterile filtered through 0.2 μm filters before being used in the C3H10T½ test.
  • FIG. 11 shows the influence of trypsin and chymotrypsin on the activity of the hh mutant: AP active fractions after a step elution of heparin-Sepharose were adjusted to a protein concentration of 0.46 mg/ml in 10 mM Na phosphate, 0.05% Tween®80 and admixed with trypsin or chymotrypsin at a protease/protein ratio (w/w) of 1:100 (A), 1:500 (B), 1:2500 (C) and 1:10000 (D). The samples were incubated for 11 h at RT. The digestion was stopped by adding aprotinin in a 5-fold weight excess and the samples were analysed in SDS-PAGE (A:) and in the C3H10T½ test (B:). 1, test mixture; 2, control without protease; 3, samples treated with trypsin; 4, samples treated with chymotrypsin; 5, control trypsin (1:100) and aprotinin at t=0; 6, control chymotrypsin (1:100) and aprotinin at t=0.
  • FIG. 12 is an elution diagram of the purification of dialysed active fractions of the hydroxylapatite column with a 1.7 ml Poros HS/M column. The UV-detected elution diagram is shown as a line and the activities of the fractions in the cell test are shown as bars.
  • FIGS. 13A and 13B are elution diagrams of the separation of the active fractions of the Poros HS/M column by means of RP-HPLC.A.: Elution diagram of 10 to 75 min. detected at 220 nm. B: Elution diagram of 15 to 40 min. detected at 280 nm.
  • FIG. 14 is an elution diagram of the purification of the dialysed active fractions of the hydroxylapatite column with a 6 ml Poros Q column. The UV signal of the elution is shown as a line, the activity in the cell test as bars.
  • FIG. 15 shows the stability of the alkaline phosphatase inducing activity towards dithiothreitol.
  • FIG. 16 shows the stability of the alkaline phosphatase inducing activity towards hydroxylamine.
  • FIG. 17 shows MALDI mass spectra of the active modified hh derivative after RP-HPLC. A, B: Spectra obtained with different laser energies and from different spots.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides isolated highly active hedgehog proteins having a molecular weight of from about 19 to about 26 kD which are esterified with a fatty acid having from 14 to 20 carbon atoms at the N-terminal domain of the protein. Esterification of the hedgehog protein with a C16 fatty acid is preferred.
  • In accordance with the present invention, the highly active hh proteins are particularly useful for inducing or stimulating chondrocytes and osteocytes as well as for treating neurodegenerative diseases. Thus, the highly active hh proteins of the present invention are useful therapeutic agents for treating bone disorders such as, for example, osteoporosis and bone fractures.
  • The method of the present invention for producing the highly active hedgehog proteins esterified with a fatty acid having from 14 to 20 carbon atoms comprises (a) providing an insect cell containing a baculovirus vector having a gene inserted therein capable of expressing a hedgehog protein, and a fermentation medium such that the insect cell and medium are capable upon fermentation of producing a fatty acid having from 14 to 20 carbon atoms; (b) fermenting the insect cell in the medium for a period of about 30 hours or less to produce the fatty acid and express the hedgehog protein esterified with the fatty acid; and (c) isolating the esterified hedgehog protein from the protein produced during the fermentation.
  • Any fermentation conditions which allow the esterification of the hedgehog protein with a fatty acid having from 14 to 20 carbon atoms can be utilized. Preferably, the fermentation period is from about 24 to about 27 hours. Generally, the fermentation is carried out at from about 10 to 35° C. and pH of about 4 to 8. Fermentation at room temperature and neutral pH is preferred. Any conventional medium for fermentation of the baculovirus expression system that allows the esterification of the hh protein can be utilized. A preferred fermentation medium is Excell 400 medium (JHR, Inc.).
  • Isolation of the esterified hh protein can be obtained by conventional techniques for isolation of proteins such as through binding of the hh protein to heparin-Sepharose and hydroxylapatite. The hedgehog proteins of the present invention can be further purified by techniques such as anion or cation exchange chromatography and reverse-phase HPLC. Purification of the cell supernatant is preferably performed in the presence of a protease inhibitor and a non-ionic detergent.
  • Hedgehog proteins are known and any hedgehog protein can be modified in accordance with this invention to esterify the protein at the N-terminal domain with a fatty acid having from 14 to 20 carbon atoms and in so doing provide the highly active hh proteins of the present invention.
  • The esterified hedgehog proteins of the present invention have the following characteristics:
      • exhibit an apparent molecular weight of 22±2 kDa under alkylating conditions in SDS-PAGE,
      • exhibit an apparent molecular weight of 24±2 kD under reducing conditions in SDS-PAGE,
      • are stabilized with respect to its activity by suramin
      • are inactivated when 8 or more amino acids are cleaved N-terminally
      • are inactivated by 90% or more when incubated with 10 mmol/l 1.4 dithioerythritol (DTE) for 2.5 hours at preferably pH 8 and 37° C.,
      • induce an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,
      • are not modified by cholesterol (C-terminal) and
      • have at least 50-fold activity compared to the recombinant hh protein isolated from the cytoplasm of E. coli. Activity within the sense of the invention is understood as the activity of alkaline phosphatase which the polypeptide can induce in mammalian cells (activity in the alkaline phosphatase test). In this method a mouse fibroblast cell line is cultured in a medium which contains foetal calf serum. Subsequently sterile filtered sample is added, the cells are lysed after ca. 5 days and alkaline phosphatase is determined in the cell lysate by means of the cleavage of a chromogenic substrate (pNP, p-nitrophenol) (J. Asahina, Exp. Cell. Res. 222 (1996) 38-47 and T. Nakamura (1997)).
  • A baculovirus expression system is understood as an expression system composed of a baculovirus vector and an insect cell as the host cell. Such expression systems are known to a person skilled in the art and are for example described by Bumcrot (1995) for hh proteins. Preferred insect cells for use in obtaining the esterified hedgehog proteins of the present invention are High five cells.
  • A hedgehog protein is understood by the invention as a secreted signal protein which is responsible for the formation of numerous structures in embryogenesis. Sonic, indian or desert hh proteins are particularly preferably used (M. Fietz et al. (1994). A hh protein with a sequence as described in the EMBL database under the No. L38518 is preferably used. Proteins of the hedgehog family exhibit a pronounced homology in their amino acid sequence which is why it is also preferable to express those nucleic acids which code for hedgehog proteins which are 80% or more homologous with the above-mentioned sequence of sonic hedgehog protein.
  • The human sonic hedgehog precursor protein is composed of the amino acids 1-462 of the sequence described in the EMBL database under No. L38518. The amino acids 1-23 represent the signal peptide, the amino acids 24-197 represent mature signal domain, the amino acids 32-197 represent the signal domain shortened by 8 amino acids and the amino acids 198-462 represent the auto-processing domain after autoproteolytic cleavage. Thus, amino acids 24-197 of the human sonic hedgehog protein described in EMBL database sequence No. L38518 represent the N terminal domain of this sequence.
  • The first 8 amino acids of the hedgehog proteins of the present invention are the first 8 amino acids of the processed hedgehog protein, for example, the amino acids Cys24-Gly31 of the sequence described in the EMBL database under No. L38518 for sonic hedgehog protein. Since the modified group can be cleaved with the first N-terminal amino acids of hedgehog protein and the activity is greatly reduced by incubation with hydroxylamine or DTE, the binding of the group is localized on these amino acids preferably in the form of a thioester, preferably as palmitic acid thioester, on the cysteine which is present in the first eight amino acids of the hedgehog protein.
  • Surprisingly when preferably the N-terminal domain of hedgehog protein is produced recombinantly in the baculovirus expression system, a highly active form of the protein (activity increased by at least 10-fold, preferably at least 100-fold compared to recombinant shh from the cytoplasm of E. coli) accumulates in the initial period of the fermentation. However, overall the amount of highly active hh mutant according to the invention is only about 0.2-5% of the total protein in the supernatant of the cells after expression in the baculovirus expression system. This mutant of the polypeptide according to the invention can be in particular isolated when the fermentation is terminated at the latest after about. 30 hours or less, preferably after about 24-27 hours. This is also surprising since a fermentation period after infection of at least 2 days has been previously described for the production of hh proteins in the baculovirus expression system (Bumcrot et al., Mol. Cell. Biol. 15 (1995) 2294-2303). It has also been described for other proteins which are produced in the baculovirus system such as rhodopsin kinase (Cha et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10577-10582) that a maximum of protein and activity is achieved after 64-88 h. According to the invention it was found for hedgehog proteins that although the amount of hedgehog protein in the fermentation supernatant greatly increases in the period between 33 and 72 hours, mainly hh protein with an activity that is known from the prior art is formed in this period. In contrast the amount of such a hh protein is considerably less (at least 3-5-fold) when the fermentation period is reduced to below ca. 30 h. which allows the identification and isolation of the highly active hh protein mutant according to the invention.
  • The molecular weight of the mutant according to the invention is 19,796.7±2 D when analysed by means of MALDI mass spectroscopy and is increased by 236.7±2 D compared to unmodified hh protein (cytoplasmically expressed hh protein in E. coli), corresponding to the molecular weight of a palmitic acid thioester. The hydrophobic modification increases the mobility in SDS-PAGE by increased SDS binding so that apparently a lower molecular weight is seen under alkylating conditions (derivatized hh protein) than under strongly reducing conditions (hh protein without derivatization). The accuracy of the molecular weight determination in SDS-PAGE is also about ±1-2 D.
  • After purification with heparin-Sepharose, hydroxyl-apatite and porous HSM ion exchanger chromatography, the hh mutant according to the invention has an activity measured via the induction of alkaline phosphatase in a cell test (activity in the AP cell test) which is increased by at least 50-fold, preferably by at least 100-fold and particularly preferably by at least 103 to 106-fold compared to soluble hh protein expressed in the cytoplasm of E. coli. Such an active hh mutant is not modified by cholesterol like the N-terminal hh fragment described by J. A. Porter since only the N-terminal and not also the C-terminal autoprocessing domain was expressed. The hh mutant according to the invention is present in a biologically active three-dimensional structure. Consequently the invention for the first time enables the isolation of highly active hedgehog protein and provides a general, reproducible method for the production and characterization of highly active hedgehog proteins.
  • Hence the invention concerns hedgehog proteins with an at least 100-fold, preferably at least 103 to 106-fold increased activity compared to the corresponding hedgehog protein which was isolated from the cytoplasm of E. coli were the activity is determined by the induction of alkaline phosphatase in the cell test.
  • A corresponding hedgehog protein which is produced in the cytoplasm of E. coli is understood according to the invention as a hedgehog protein which is isolated in a soluble form in the cytoplasm after expression in E. coli. In this process a vector is used as the expression vector which contains a nucleic acid to be expressed which codes for a hedgehog protein of the same amino acid sequence as the nucleic acid to be expressed of the expression vector which is used for expression in the baculovirus expression system. However, in this process it may be expedient to change one or other amino acid in the baculovector or E. coli vector in order to for example improve the expression or the solubility. However, for the comparison of activities of the hedgehog protein according to the invention with the E. coli protein it is expedient to use expression vectors for identical proteins. The hh protein that is formed in this process is not post-translationally modified (no derivatization with cholesterol etc.).
  • The hh mutant according to the invention is very sensitive towards proteases which is why it is preferable to add protease inhibitors such as for example aprotinin, EDTA (up to 1 mmol/l), PMSF or pepstatin or a mixture thereof to the supernatant of the fermentation.
  • Furthermore it is preferable to add non-ionic detergents such as polysorbate (e.g. Tween®20, Tween®80, Triton®X100) during the purification, preferably before or after the first crude purification over heparin-Sepharose. Since this also stabilizes the hh proteins according to the invention.
  • In a first step for the purification of the protein according to the invention it is expedient to carry out a chromatography on heparin-Sepharose. It is preferable to carry out this chromatography as a step elution i.e. preferably to elute at a concentration of at least 0.7 mol/l NaCl (preferably 1.2 mol/l) after washing with 250 mmol/l NaCl.
  • It is particularly preferable to carry out a hydroxylapatite chromatography to purify the hh mutant according to the invention. This achieves a good concentration of the activity with relatively low losses (<50%). Further suitable chromatographic steps are for example a heparin-Sepharose chromatography (Miao et al., J. Neurosci. 17 (1997) 5891-5899) which is, however, preferably carried out in the presence of non-ionic detergents. Furthermore it is preferable to carry out a dialysis after the heparin-Sepharose chromatography in which it is particularly preferable that in this dialysis the pH value of the buffer is at least pH 5, in particular pH 6.5-7.5 and the ionic strength of the buffer corresponds to a solution of 1-20 mM sodium phosphate and 10-100 mM NaCl in particular 50 mM NaCl and the dialysis is carried out at a low concentration of total protein (1 mg/ml or less, preferably 0.5 mg/ml or less).
  • Furthermore, it is also preferable to add suramin during the purification or at least before determining the activity of the protein. The activity can also be stabilized by adding serum albumin (at least 50 μg/ml to 5 mg/ml) to the sample before dilution in the cell test. This also stabilizes the activity. In the case of suramin it was previously only known that it is suitable for detaching hh proteins from the cell surface or the extracellular matrix (Bumcrot et al., see above).
  • Furthermore, it is known that suramin inhibits the activity of growth factors (Middaugh et al., Biochem. 31 (1992) 9016-9024). Surprisingly it was found that the activity of hh proteins is increased by adding suramin.
  • For the further purification it is preferable to repeat chromatography on heparin-Sepharose and hydroxylapatite.
  • For the further purification it is additonally preferable to carry out an ion exchange chromatography with porous HS/M and/or Poros-Q (Boehringer Mannheim GmbH, DE) and preferably subsequently a RP-HPLC. In contrast to other ion exchanger media, only low losses of activity and a good separation of the active hh form is observed with Poros exchangers.
  • In a further embodiment of the invention the hh mutant according to the invention can be used to produce a pharmaceutical composition which is also a subject matter of the invention. This pharmaceutical composition contains a pharmacologically effective dose of the protein according to the invention and can be administered systemically as well as locally. It is also preferable to use the proteins according to the invention in combination wish other proteins of the hedgehog family or bone growth factors such as bone morphogenetic proteins (BMPs), (Wozney et al., Cell. Mol. Biol. of Bone, Bone Morphogenetic Proteins and their Gene Expression, 131-167, Academic Press Inc. 1993) or parathyroid hormones (Karablis et al., Genes and Development 8 (1994) 277-289).
  • The protein according to the invention can be used advantageously to induce or stimulate chondrocytes and osteocytes in an osteoinductive pharmaceutical composition as well as to treat neurodegenerative diseases. Osteoinductive pharmaceutical compositions are for example known from the U.S. Pat. No. 5,364,839, WO 97/35607, WO 95/16035.
  • When the protein according to the invention is administered locally it is preferable to use it in combination with a suitable matrix as a carrier and/or with a sequestering agent. Such a matrix is suitable for slowly releasing the protein in vivo in an active form in particular in the vicinity of bone and cartilage. The sequestering agent is a substance which facilitates administration for example by injection and/or prevents or at least delays migration of the protein according to the invention from the site of administration.
  • A biocompatible degradable material for example based on collagen or other polymers based on polylactic acid, polyglycolic acid or co-polymers of lactic acid and glycolic acid are particularly suitable as a matrix material. Such polymer matrices are described for example in WO 93/00050.
  • Sequestering agents are for example cellulose and cellulose-like materials and for example alkyl cellulose, carboxymethyl cellulose, hyaluronic acid, sodium alginate, polyethylene glycol and polyvinyl alcohol of which hyaluronic acid is particularly preferred especially in a pharmaceutical composition even without carrier matrix.
  • It is also preferable for the production of the pharmaceutical composition to add auxiliary substances such as mannitol, sucrose, lactose, glucose or glycine and antioxidants such as EDTA, vitamin C, citrate and detergents, preferably non-ionic detergents like polysorbates and polyoxyethylenes. A pharmaceutical composition which is buffered in the pH range 4-8 is also preferred.
  • In a further preferred embodiment a pharmaceutical composition of the hedgehog protein according to the invention together with suramin and/or serum albumin is preferred and this is advantageously used.
  • The following example, publications and figures further elucidate the invention, the protective scope of which results from the patent claims. The described methods are to be understood as examples which still describe the subject matter of the invention even after modifications.
  • EXAMPLE 1 Expression of Recombinant Human Sonic hh (shh)
  • The N-terminal domain of human shh with the amino acids 24-197 (EMBL accession No. L 38518) was expressed with the N-terminal signal peptide 1-23 as described by Miao (J. Neurosci. (1997) 17, 5891-5899) and Bumcrot et al., (Mol. Cell. Biol. (1995) 15, 2294-2303) for the rat protein by means of recombinant baculovirus in High Five cells (Invitrogen, Leek, N L, Order No. E 855-02) using Excell 400 medium (JHR, Inc.) in which sufficient virus was used to infect each cell on average with one virus (multiplicity of infection (m.o.i.):1).
  • The fermenter contents were clarified after 26 or 72 h by centrifugation at 1000 g and filtration and the supernatant or the permeate was stored at -80° C. until further use. Fermentation samples were analysed for their content of alkaline phosphatase inducing activity [Nakamura et al. (1997), Kinto et al. (1997) FEBS Lett. 404, 319-323] and for their content of shh protein by means of RP-HPLC (Vydac C18, gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid, TFA) or SDS-PAGE.
  • In the preferred process the fermentation was terminated after 24-32 h (preferably after 24-27 h) fermentation time and the supernatant was clarified.
  • EXAMPLE 2 Purification of the Active hh Mutant on heparin-Sepharose and hydroxyapatite
  • 1 Tablet of “complete” inhibitor mix (Boehringer Mannheim GmbH, order No. 1873580) was added per 50 ml supernatant to the clarified supernatant after thawing and 3.5 l of this solution was applied at 4° C. to a heparin-Sepharose column (volume 90 ml; Pharmacia Biotech) which had previously been equilibrated with 20 mM sodium phosphate, pH 7.2. After the sample application it was washed with 20 mM sodium phosphate, 0.05% Tween®80, pH 7.2 (=buffer A) and unspecifically bound protein was eluted by a wash step with buffer A which additionally contained 0.25 M NaCl. The activity was obtained by a subsequently elution with buffer A which additionally contained 1.2 M NaCl.
  • This eluate was subsequently diluted with one volume 10 mM sodium phosphate, 0.05 % Tween® 80, 50 mM NaCl, pH 7.2 (=buffer B) and dialysed against buffer B at 4° C.
  • The dialysate was applied to a hydroxyapatite column (volume 10 ml; Makro Prep; 40 μm, type I; BIO-Rad) equilibrated with buffer B. It was eluted with a gradient of 10 to 300 mM NaP in buffer B (2×200 ml).
  • Aliquots of the fractions were analysed for their ability to stimulate alkaline phosphatase in a mouse fibroblast cell line e.g. C3H10T½ cells as well as by means of SDS-PAGE and RP-HPLC. The remainder of the fractions was stored at −80° C. until further processing. The maximum activity elutes at the end of the gradient between 0.25-0.3 M sodium phosphate whereas inactive or only weakly active forms of shh already elute much earlier from the column.
  • The active fractions were pooled and dialysed against buffer B at 4° C. and applied to a 1 ml HiTrap heparin column (Pharmacia Biotech) which had been equilibrated with 20 mM potassium phosphate, 0.05 Tween®80, pH 7.2. It was eluted by a gradient of 20-1400 mM KCl in 20 mM potassium phosphate, 0.05 % Tween® 80, 50 mM NaCl, pH 7.2. Active fractions were identified by the stimulation of alkaline phosphatase in C3H10T½ cells, and alkylated and reduced samples were analysed by means of SDS-PAGE and Western blot with an antibody against the N-terminus of shh.
  • EXAMPLE 3 Purification of the Active hh Mutant on a Cation Exchanger and RP.HPLC
  • (1) Purification by cation exchange chromatography on PorosHS/M:
  • The active fractions after the hydroxylapatite or HiTrap heparin chromatography of example 1 were pooled and dialysed against 50 mM potassium phosphate, 0.05% Tween®80, pH 7.2 (=buffer C) and applied to a 1.7 ml Poros®HS/M column (Boehringer Mannheim GmbH, GER) which had been equilibrated in buffer C. It was eluted by a gradient of 40 column volumes of 50-1000 mM KCl in buffer C and at a flow rate of 3 ml/min. Active fractions were identified by stimulation of alkaline phosphatase in C3H10T½ cells. The active fractions eluted at a salt concentration of ca. 400-700 mM KCl and the alkylated samples had a purity of ca. 50%; in the SDS-PAGE under alkylating conditions whereas the major portion of the proteins already eluted at a salt concentration of ca. 80-400 mM KCl. It is possible to identify the highly active hh derivative contained in these fractions by subsequent RP-HPLC and mass analysis of the elution peak or by an appropriate analysis of the gel bands in the SDS-PAGE with a molecular weight of ca. 22 kD.
  • (2) Purification by RP-HPLC:
  • Active fractions of the Poros®-HS/M chromatography were further purified by RP-HPLC. For this 3.2 ml of a highly active fraction was applied to a 2.1×150 mm butyl column (Vydac™ 214TP5215) which had been equilibrated in 20% acetonitrile, 0.1% trifluoroacetic acid (TFA). It was eluted at 25° C. in a gradient of 20-90% acetonitrile in 0.1% TFA and analysed by detecting the absorbance at 220 nm and 280 nm. In comparison to weakly active unmodified monomeric or dimeric hh forms, the concentrated highly active hh derivative did not elute until a somewhat higher concentration of acetonitrile (ca. 41.2%). This species was collected and its mass was determined by MALDI mass spectrometry.
  • (3) Mass Analysis of the Active hh Derivative After RP-HPLC
  • For the mass analysis the RP eluate described above (total volume 200 μl) was admixed with 5 μl 25 mM sinapinic acid in 30% (w/w) acetonitrile/70% water/0.1% trifluoro-acetic acid, evaporated to dryness in a Speedvac concentrator and dissolved in 5 μl 30% (v/v) acetonitrile/70% water/0.1% trifluoroacetic acid. 1 μl of the solution obtained in this manner (referred to as solution A in the following) was mixed with 1 μl 25 mM sinapinic acid in 30% (v/v) acetonitrile/70% water/0.1% trifluoroacetic acid, applied to the target, dried in laboratory air and after drying it was measured in a Bruker REFLEX MALDI mass spectrometer with a so-called delayed extraction source. Only one molecular species was detected in the mass spectrum obtained in this manner apart from alkali and matrix adducts. Since the determination of the molecular mass without using an internal standard is only ±0.03%, the mass spectrum of an aliquot of solution A to which a protein of a known mass had been added was measured in addition to the mass spectrum of the pure solution A. For this 0.5 μl of solution A, 0.5 μl of an equivalently prepared solution of a shortened hedgehog molecule with an average molecular weight of 18900.1 D and 1 μl 25 mM sinapinic acid in 30% (v/v) acetonitrile/70%. water/0.1% trifluoroacetic acid were mixed, applied to the target, dried in laboratory air and after drying were measured by mass spectrometry in the same manner. The spectra obtained in this manner (FIG. 17) were calibrated with the aid of singly and doubly charged ions of the molecule used for spiking. The molecular masses determined for the active hedgehog species present in solution A are summarized in the following Table 1.
    TABLE 1
    Difference from the
    Molecular mass of molecular mass of the
    Spectrum of the active hedgehog unmodified hedgehog
    FIG. 1/signal molecule molecule (19560.0 D)
    A/MH+ 19795.6 D 235.6 D
    A/MH++ 19797.6 D 237.6 D
    B/MH+ 19796.3 D 236.3 D
    B/MH++ 19797.2 D 237.2 D
  • Taking into consideration the inaccuracy of the mass determination of ±0.01%, the difference between the molecular mass determined for the active hedgehog molecule and the molecular mass of the unmodified hedgehog molecule is 236.7±2 D. Among the known naturally occurring covalent protein modifications only esterification with C16 fatty acids (palimitic acid: mass increase by 238.4 D, monounsaturated palmitic acids: mass increase by 236.4 D) leads to an increase of the molecular mass which is compatible with the found increase in mass (Turner and Smith, Molecular Biotechnol. 8 (1997) 233-249).
  • (4) Purification by Anion Exchange Chromatography on Poros®-Q:
  • In addition or alternatively to purification by means of cation exchange chromatography on Poros®-HS/M, it is also possible to purify the HA or the heparin HiTrap eluates by means of anion exchange chromatography on Poros®-Q. For this the active fractions were pooled, dialysed against 20 mM Tris/HCl, 0.05% Tween®80, pH 9.0 (=buffer D) and applied to a Poros®-Q column which had been equilibrated in buffer D. It was eluted by a gradient of 60 column volumes of 0-1000 mM NaCl in buffer D. Active fractions were identified by stimulation of alkaline phosphatase in C3H10T½ cells. Active hh protein eluted at a salt content of 90-175 mM NaCl. These active fractions can be further purified and characterized by means of Poros®-HS/M or RP-HPLC or SDS-PAGE and Western blot as described above.
  • EXAMPLE 4
  • Stability of the Active hh Mutant Towards Reducing Agents
  • (1) Stability Towards dithiothreitol (DTT)
  • The step eluate of a heparin-Sepharose column containing buffer A which additonally contained 1.2 M NaCl (see example 3) was diluted with 1 volume 0.05 % Tween® 80 and 1/10 vol. 1 M Tris/HCl, pH 8 was added for the samples at pH 8. DTT was added to the samples at final concentrations of 0 mM, 1 mM, 10 mM and 50 mM. After 2 h incubation at 37° C., each of the samples was admixed with 1/10 volumes (50 μl) 10 mg/ml BSA and dialysed against PBS. Before use in the C3H10T½ cell test, a final concentration of 0.1 mg/ml suramin was added to each sample in order to stabilize the remaining activity.
  • It turned out that the activity at pH 7.2 is stable up to a concentration of 10 mM DTT, however, at pH 8 treatment with 1 mM DTT already leads to a considerable reduction of activity. Such a pH dependency would be expected for the reduction of sulphur in disulfide bridges as well as of thioesters of fatty acids (Issartel et al., Nature 351 (1991) 759-761).
  • (2) Stability Towards hydroxylamine (HA):
  • The step eluate of a heparin-Sepharose column with buffer A which additionally contained 1.2 M NaCl (see example 3) was adjusted with NaOH to pH 8.0 or with HCl to pH 5.5. Aliquots of these samples were each admixed with 0 mM, 66 mM, 250 mM or 1 M of NH2OH with the corresponding pH value and incubated for 14 h at RT. The samples were subsequently dialysed against 20 mM NaP, 250 mM NaCl, 0.05% Tween80, pH 7.4 and admixed with 1 mg/ml (final concentration) BSA and 0.1 mg/ml (final concentration) suramin before analysis in the cell test.
  • It turned out that the activity at pH 5.5 is stable up to 66 mM HA but treatment with 66 mM HA at pH 8 already led to a considerable reduction of activity. Such a pH dependency would be expected for the cleavage of thioesters but not for hydroxyl esters of fatty acids (Issartel et al., Nature 351 (1991) 759-761; Maggee et al., EMBO J. 4 (1985) 1137-1144).
  • EXAMPLE 5
  • Induction of Alkaline Phosphatase in the Cell Test (Determination of the Activity of Alkaline Phosphatase)
  • 5000 cells of the murine mesenchymal pluripotent line: C3H10T½ (ATCC CCl-226) were sown in each well of a 96-well microtitre plate. The cells were in 100 μl DMEM, 2 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% foetal calf serum, FCS. On the next day the active substances to be examined were added at the appropriate concentrations in a volume of 100 μl after 20-500-fold predilution in culture medium. The test was stopped after 5 days. For this purpose the supernatants were discarded and the cells were washed once with PBS. The cells were lysed in 50 μl 0.1% Triton®X-100 and frozen at −20° C. After thawing 25 μl was used for the protein determination and 25 μl for the determination of the activity of alkaline phosphatase.
  • Protein Determination According to the Instructions of the Manufacturer Pierce:
  • 75 μl distilled H2O was added to the mixture, then 100 μl BCA protein reagent was added (Pierce Micro BCA, No. 23225). After 60 min the optical density (OD) at 550 nm was measured.
  • Activity of the Alkaline Phosphatase According to the Instructions of the Manufacturer Sigma:
  • 100 μl reaction buffer (Sigma 221) was added to the preparation. A substrate capsule (Sigma 104-40) was dissolved in 10 ml redistilled H2O and then 100 μl was added to the test mixture by pipette. The OD was measured at 405 nm after the yellow coloration. In the reaction alkaline phosphatase converts p-nitrophenyl phosphate into p-nitrophenol.
  • The ODs were each converted into nmol or μg by means of standard curves. The evaluation was according to the formula:
  • nmol PNP per (measured) minute per mg (cell) protein
  • EXAMPLE 6
  • Comparison of the Specific Activities of Unmodified hShh from E. coli with the hShh Derivative Purified from BVCM
  • In order to compare the specific activities of the diverse hh forms, hh protein was used at a defined concentration in the C3H10T½ cell test. In this case the protein determination was carried out for the unmodified hh protein from E. coli by means of its UV absorbance at 280 nm (Mach, H., et al., Anal. Biochem. 200 (1992) 74-80). The concentration of the hh derivative purified from the baculovirus fermentation supernatant was determined by means of RP-HPLC. The concentration in the stock solution was determined by integrating the area under the absorbance curve of the hh peak in the RP-HPLC where the detection was carried out at 220 and 280 nm and a calibration curve was established by an analogous chromatography using stock solutions of unmodified shh of known concentration. The relative activity of the isolated shh proteins was determined in the C3H10T½ cell test by determining the stimulation of the expression of alkaline phosphatase in these cells (example 5; cell test) compared to the baculovirus fermentation supernatant (BVCM after 24 hour fermentation) in a 1:40 dilution where the individual values were corrected for the base line activity of the cells in the absence of added shh. This determination of the relative activity is preferred since the stimulatability of the cells is influenced by the medium used and the preculture of the cells.
  • Table 2 shows the relative activities of purified, unmodified shh from E. coli and of the shh derivative purified from the supernatant of the baculovirus fermentation supernatant, which had been purified by chromatography on heparin-Sepharose, hydroxylapatite and Poros HS/M.
  • As shown in Table 2, hh protein purified from E. coli must be present at a concentration of ca. 240 μg/ml but the hh protein derivative only needs to be present at a concentration of ca. 2.3 ng/ml in the cell test in order to reach the same alkaline phosphatase activity as the BVCM at a 1:40 dilution. Thus the hh protein derivative has a ca. 104-105-fold higher specific activity than the unmodified hh protein.
    TABLE 2
    Hh Hh
    concentration Dilution concentration relative AP
    of the stock in the in the cell inducing
    Hh source solution cell test test activity
    BVCM  12 μg/ml 1:40 0.3 μg/ml 1
    Poros HS/M 0.56 μg/ml   1:100 5.6 ng/ml 2.4
    purified
    hShh
    derivative
    Unmodified 860 μg/ml 1:20 43 μg/ml 0.18
    hShh monomer
    from E. coli
    Buffer control 1:40 0
  • EXAMPLE 7
  • in vivo Activity of palmitoylated hh
  • The in vivo activity of modified and unmodified shh protein was examined in an animal model established for bone growth factors (Mackie & Trechsel, Bone 11 (1990) 296; Kling, L., et al., J. Bone Min. Res. 11 (Suppl. 1) (1996) 153). Seven week old female BALB/c mice were subcutaneously injected daily over a period of 15 days with 1, 10 or 50 μg shh in a volume of 50 μl into the cranial calotte. 14 days after completion of the treatment, the calottes were removed and purified of surrounding connective tissue. Subsequently the weights of the standardized explants and the X-ray density were analysed. Modified shh exhibited a higher osteoanabolic effect than unmodified shh.
  • EXAMPLE 8
  • Influence of trypsin and chymotrypsin on the Activity of shh
  • After step elution of heparin-Sepharose the AP active fractions were admixed with trypsin or chymotrypsin at different protease/protein ratios. After incubation of the samples for 20 h at 25° C. the digestion was stopped by addition of aprotinin and the samples were analysed by means of SDS-PAGE and in the C3H10T½ test (see FIG. 11).
  • It turned out that the hedgehog protein is degraded to a shortened form which migrate at 20 kDa in the SDS-PAGE. By means of amino-terminal sequencing and MALDI mass spectrometry it could be shown that the mature hh protein is degraded by the chymotrypsin or trypsin treatment at a protease/protein ratio (w/w) of 1:100 to fragments in which the first 7 or 14 amino-terminal amino acids are absent. As shown by the-results of the C3H10T½ test in FIG. 11, the alkaline phosphatase inducing activity is also reduced when the hh protein is shortened.
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    • Kinto et al., FEBS Letters, 404 (1997) 319-323
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    • Lai, C. J. et al., Development 121 (1995) 2349
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    • Mackie & Trechsel, Bone 11 (1990) 296
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Claims (20)

1. A protein in isolated form wherein said protein comprises a human hedgehog protein which is esterified at the N-terminal domain with a fatty acid having from 14 to 20 carbon atoms, said protein having a molecular weight of from about 19 to about 26 kD.
2. The protein of claim 1, wherein the fatty acid is palmitic acid.
3. The protein of claim 2, wherein the amino acid sequence of said protein is at least 80% homologous with the amino acid sequence of EMBL database sequence No. L38518.
4. The protein of claim 3, wherein said protein contains the amino acid sequence of EMBL database sequence No. L38518.
5. The protein of claim 3, wherein the protein is esterified at a cysteine residue at the N-terminal domain of said sequence.
6. A method for producing a hedgehog protein esterified with a fatty acid having from 14 to 20 carbon atoms comprising:
a) providing an insect cell in a medium, said medium and said insect cell being capable upon fermentation of producing a fatty acid having from 14 to 20 carbon atoms, said insect cell containing a baculovirus vector having a gene inserted therein capable of expressing a hedgehog protein;
b) fermenting said insect cell in said medium for a period of about 30 hours or less to produce the fatty acid and-express the hedgehog protein esterified with said fatty acid; and
c) isolating said esterified hedgehog protein from the protein produced during said fermentation.
7. The method of claim 6,.wherein the fermentation period of said host cell is from about 24 hours to about 27 hours.
8. The method of claim 6, further comprising isolating said esterified hedgehog protein in the presence of a protease inhibitor and a non-ionic detergent.
9. The method of claim 6, further comprising isolating said esterified hedgehog protein in the presence of suramin.
10. Post-translationally processed hedgehog protein mutant which is obtainable by expressing a gene which codes for a hedgehog protein in a baculovirus expression system in a fermentation for a period of up to 30 hours, purifying the cell supernatant in the presence of a protease inhibitor and a non-ionic detergent and isolating the hh mutant which binds to heparin-Sepharose and hydroxylapatite and is characterized in that this hh mutant
exhibits a molecular weight of 22±2 kDa under alkylating conditions,
exhibits a molecular weight of 24±2 kD under reducing conditions,
is stabilized with respect to its activity by suramin
is inactivated when 8 or more amino acids are cleaved N-terminally
is inactivated by 90% or more when incubated with 10 mmol/l DTE for 2.5 hours at 37° C.,
induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,
is not modified by cholesterol and
has an at least 50-fold activity compared to the recombinant hh protein isolated from the cytoplasm of E. coli.
11. Process for the production of a post-translationally processed hedgehog protein mutant by expressing a gene which codes for a hedgehog protein in a baculovirus expression system in a fermentation for a period of 24 to 27 hours, purifying the cell supernatant in the presence of a protease inhibitor and a non-ionic detergent and isolating the hh mutant which binds to heparin-Sepharose and hydroxylapatite and characterized in that this hh mutant
exhibits a molecular weight of 22±2 kDa under alkylating conditions,
exhibits a molecular weight of 24±2 kD under reducing conditions,
is stabilized with respect to its activity by suramin
is inactivated when 8 or more amino acids are cleaved N-terminally
is inactivated by 90% or more when incubated with 10 mmol/l DTE for 2.5 hours at 37° C.,
induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,
is not modified by cholesterol and
has an at least 50-fold activity compared to the recombinant hh protein isolated from the cytoplasm of E. coli.
12. Process as claimed in claim 11, wherein, after chromatography on heparin-Sepharose, it is dialysed against lower ionic strengths.
13. Process as claimed in claim 12, wherein the dialysis is carried out in the presence of 10-100 mmol/l sodium chloride.
14. Pharmaceutical composition containing a hh mutant as claimed in claim 10.
15. Pharmaceutical composition as claimed in claim 14, containing suramin, serum-albumin, a biocompatible matrix and/or a sequestering agent.
16. Process for the production of a pharmaceutical composition by combination of a hh mutant as claimed in claim 10 with a pharmaceutical auxiliary substance or with suramin.
17. Process for the production of a pharmaceutical composition by combination of a hh mutant as claimed in claim 10 with a biocompatible matrix and/or a sequestering agent.
18. Post-translationally processed hedgehog protein with an at least 50-fold higher activity than the hedgehog protein expressed cytoplasmically in E. coli.
19. Pharmaceutical composition containing a hedgehog protein as claimed in claim 18.
20. Process for the production of a pharmaceutical composition, wherein a hedgehog protein as claimed in claim 18 is used as an essential component of this pharmaceutical composition.
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CA2312009A1 (en) 1999-06-10
US6994990B1 (en) 2006-02-07
AU752816B2 (en) 2002-10-03
WO1999028454A1 (en) 1999-06-10
EP1034269A1 (en) 2000-09-13
MA26570A1 (en) 2004-12-20
IL136230A0 (en) 2001-05-20
US20040138130A1 (en) 2004-07-15
AU1757099A (en) 1999-06-16
JP2001525341A (en) 2001-12-11
IL136230A (en) 2007-08-19

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