[go: up one dir, main page]

WO1999000417A1 - Procede d'obtention de proteines fixant l'heparine dans des cellules de mammiferes - Google Patents

Procede d'obtention de proteines fixant l'heparine dans des cellules de mammiferes Download PDF

Info

Publication number
WO1999000417A1
WO1999000417A1 PCT/DK1998/000275 DK9800275W WO9900417A1 WO 1999000417 A1 WO1999000417 A1 WO 1999000417A1 DK 9800275 W DK9800275 W DK 9800275W WO 9900417 A1 WO9900417 A1 WO 9900417A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid sequence
hbp
nucleic acid
seq
set forth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/DK1998/000275
Other languages
English (en)
Inventor
Ivan Svendsen
Hans Jakob Flodgaard
Poul Baad Rasmussen
Finn C. Wiberg
Søren E. BJØRN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Priority to AU80124/98A priority Critical patent/AU8012498A/en
Publication of WO1999000417A1 publication Critical patent/WO1999000417A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins

Definitions

  • the invention is directed to a method for the production of heparin-binding proteins in recombinant mammalian cells, particularly eukaryotic cells producing an acidic proteoglycan and hybrid mammalian cells.
  • the invention is further related to said recombinant mammalian cells, as well as methods for producing said cells.
  • the proteins have been named human heparin-binding protein (hHBP) and porcine heparin-binding protein (pHBP), respectively, owing to their high affinity for heparin;
  • hHBP human heparin-binding protein
  • pHBP porcine heparin-binding protein
  • CAP37 protein cationic antimicrobial protein due to its antimicrobial activity.
  • the protein has been found to regulate monocyte/macrophage functions such as chemotaxis, increased survival, and differentiation (reviewed in Pereira, 1995, J. Leuk. Biol. 57:805-812, also see U.S. Patent Nos. 5,458,874 and 5,484,885).
  • HBP has been shown to mediate detachment and contraction of endothelial cells and fibroblasts when added to such cells grown in monolayer culture. HBP also stimulates monocyte survival and thrombospondin secretion (E. ⁇ stergaard and H. Flodgaard, 1992, J. Leukocyte Biol. 51:316 ff). A protein with the first 20 N-terminal amino acid residues identical to those of hHBP and CAP37 called azurocidin has also been isolated from the azurophil granules (J.E. Gabay et al, 1989, Proc. Natl. Acad. Sci. USA 86:5610 ff.; C.G.
  • HBP The structure of HBP appears from WO 89/08666 and H. Flodgaard et al., op. cit. HBP has otherwise been termed CAP37 (cf. WO 91/00907, U.S. Patent Nos. 5,458,874 and 5,484,885) and azurocidin (cf. C.G. Wilde et al., J. Biol. Chem. 265, 1990, p. 2038).
  • HBP has previously been isolated from neutrophil leukocytes (Flodgaard et al., 1991, Eur. J. Biochem. 197:535-547). However, the yields have been very low. Furthermore, HBP has been produced via recombinant DNA methods in insect cells (Rasmussen et al., 1996, FEBS Lett. 390:109-112). However, it has been found that the insect cell system cannot produce mature HBP. Instead, the product obtained contains the seven amino acid residue N- terminal prosequence. This molecule is inactive in animal models. In order to produce active mature HBP in the insect cell system, a prosequence deleted DNA construct has to be used. This system only produces modest amounts of HBP. A deleted DNA construct interferes with the processing machinery and therefore considerable amounts (20%) of truncated inactive material is produced. These inactive forms have to be eliminated and consequently the yield is lowered and the purification procedure is complicated.
  • Recombinant HBP (referred in the reference as CAP37) has also been produced in the human kidney 293 cell line ( R. Alberdi et al, 1997 FASEB J. 11:1915-1915).
  • An RSV-PL4 expression vector was used. This vector contained a transferrin signal peptide for secretion, the HPC4 epitope for immunoaffinity purification, a factor Xa cleavage site, and a neomycin- resistant gene for G418 selection.
  • Functional heparin-binding protein could only be produced by cleaving the recombinant protein in vitro at the Factor Xa cleavage site using bovine factor Xa to separate the fusion peptide from the recombinant heparin-binding protein.
  • basophils In contrast to mast cells, basophils have polylobed nuclei with a condensed nuclear chromatin pattern, like that of other granulocytes. Furthermore, basophils are usually smaller and contain fewer granules than mast cells of the corresponding species. After differentiation and maturation in the bone marrow, basophils like other granulocytes circulate in the blood. They are not generally detected in extravascular tissues. Like mast cells, basophils contain metachromatic cytoplasmic granules, store histamine and express IgE receptors. However, it appears that basophils do not retain a capacity for cell division or transdifferentiation into mast cells.
  • Basophils have been found to produce proteoglycans with glycosaminoglycan (GAG) side chains (reviewed in L. Enerback, The Mast Cell System, in HEPARIN, Chemical and Biological Properties, Clinical Applications pp 97-113, Eds. D.A. Lane and U. Lindahl, Edward Arnold, A division of Hodder & Stoughton, London, Melbourne Auckland 1989).
  • GAG glycosaminoglycan
  • RBL-1 cells have been found to produce predominantly over sulfated chondroitin and heparin-like GAGs (Seldin et al., 1984, J. Biol Chem. 260:11131-11139). Such substances could potentially prevent certain proteins such as HBP from exerting its biological activity intracellularly.
  • HBP HBP-binding protein
  • sulfated heparin The activity of HBP is dependent on a cluster of positive charges on one side of the molecule. Neutralization of this site with sulfated heparin abrogates its chemotactic activity (G ⁇ ricke and Flodgaard, 1994, J. Cell. Biochem., Keystone Symposia on Molecular & Cellular Biology, Supplements 18 A, January 4-23, 1994 Abstr.
  • Heterologous proteins have been expressed in one type of basophilic cell, rat basophilic leukemia cells (RBL-1). Specifically, human cathepsin G transfected to rat RBL-1 cells has been shown to be proteolytically processed into enzymatically active forms and to be transferred to granular organelles. (Gulberg et al., 1994, J. Biol. Chem. 269:25219-25225).
  • proteinase 3 (PR3), a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors, when transfected to RBL cells, has been found to be synthesized as a 29 kD protein core glycosylated on two distinct sites (Garwicz et al., 1997, J. Leuk. Biol. 61 : 1-11). Furthermore, with respect to PR3 expressed in RBL cells, oligosaccharide trimming and proteolytic processing was found to occur and the protein was targeted for granular storage in a form antigenic for anti-neutrophil cytoplasmic autoantibodies.
  • PR3 proteinase 3
  • HBP heparin-binding protein
  • the invention is therefore directed to a method for producing a mature heparin-binding protein (HBP) comprising (a) culturing a recombinant eukaryotic cell expressing an acidic proteoglycan(s) or hybrid mammalian cell comprising a nucleic acid sequence encoding heparin-binding protein (HBP) under conditions conducive to expression of the polypeptide; and (b) recovering said mature heparin-binding protein.
  • the recombinant eukaryotic cell expressing acidic proteoglycan or hybrid mammalian cell may be obtained by introducing a nucleic acid sequence encoding said heparin-binding protein into said cell.
  • the invention is further directed to said recombinant eukaryotic cell expressing an acidic proteoglycan(s) and recombinant hybrid mammalian cells.
  • the hybrid mammalian cell is a fused myeloma cell and cell expressing an acidic proteoglycan.
  • Figures 1 A and IB respectively show the constructs used, pcDNA3-HBP and pcDNA ⁇ pro.
  • Figure 2 shows Coomassie-stained SDS-PAGE (8-15%) comparing different HBP forms.
  • Lane 1 recombinant HBP obtained from RBL-lcells.
  • Lane 2 recombinant HBP obtained from insect cells.
  • Figure 3 shows a peptide map of human HBP (top), RBL-1 HBP (middle) and insect cell HBP (bottom).
  • Figure 4 shows a carbohydrate map of the three HBP samples after release and 2-AB labelling of the carbohydrates.
  • CarboPac PA- 100 column sodium acetate gradient at high pH, fluorescence detection.
  • Figure 5 shows a carbohydrate map of the three HBP samples after release, 2-AB labelling and desialylation of the carbohydrates. Conditions as in Figure 4, except the gradient is modified.
  • Figure 6 shows size-exclusion chromatography (SEC) of the three HBP samples after release, 2-AB labelling and desialylation of the carbohydrates.
  • Top panel Human HBP
  • Middle panel RBL-1 HBP
  • Bottom panel Insect cell HBP.
  • Figure 7 shows MALDI-MS of the three HBP samples.
  • Top panel Human HBP
  • Middle panel RBL-1 HBP
  • Bottom panel Insect cell HBP.
  • Figure 8 shows CE of HBP samples in 25 mM ammonium phosphate, 0.05% polyvinyl alcohol, pH 2.5.
  • Capillary fused silica with Z-cell (LC Packings), ID 75 Am, L 45 cm, I 37.5 cm.
  • Inj. 10 sec.
  • Voltage 11 kV -31 pA.
  • Detection 214 nm.
  • Figure 9 shows CE of HBP samples in 25 mM ammonium phosphate, 0.005% polyarginine, pH 6 .5.
  • Capillary fused silica with Z-cell (LC Packings), ID 75 Am, L 45 cm, 137.5 cm.
  • Inj. 20 sec.
  • Voltage 11 kV -23 FA. Reversed polarity. Detection: 214 nm.
  • Figure 10 shows cation-exchange HPLC of the three HBP samples.
  • Figure 11 GP-HPLC of the three HBP samples.
  • the HBP may suitably be of mammalian, in particular human or porcine, origin.
  • the HBP is human HBP which has at least about an 80% identity with the amino acid sequence set forth in SEQ ID NO:l, which includes the human HBP signal sequence, pro sequence, and sequence of the mature human HBP; SEQ ID NO:3, which includes the pro sequence and sequences of the mature protein; and SEQ ID NO:5, which includes the sequence of the mature human HBP, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97% (hereinafter "homologous polypeptides”), which qualitative retain the activity of said heparin-binding protein.
  • homologous polypeptides which qualitative retain the activity of said heparin-binding protein.
  • the HBP is porcine HBP which has at least about an 80% identity with the amino acid sequence set forth in SEQ ID NO: 7, which includes the porcine HBP signal sequence, pro sequence, and sequence of the mature porcine HBP; SEQ ID NO:9, which includes the pro sequence and sequences of the mature protein; and SEQ ID NO:l l, which includes the sequence of the mature porcine HBP, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%, which qualitative retain the activity of said heparin-binding protein.
  • SEQ ID NO: 7 which includes the porcine HBP signal sequence, pro sequence, and sequence of the mature porcine HBP
  • SEQ ID NO:9 which includes the pro sequence and sequences of the mature protein
  • SEQ ID NO:l l which includes the sequence of the mature porcine HBP, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%, which qualitative retain the activity of said heparin-binding
  • GGCTCCAGCCCCC TTTTGGAC ATCGTTGGCGGC CGGAAGGCGA GGCCCCGCCA GTTCCCGTTC CTGGCCTCCA TTCAGAATCA AGGCAGGCAC TTCTGCGGGG GTGCCCTGAT CCATGCCCGCTTCGTGATGA CCGCGGCCAG CTGCTTCCAA AGCCAGAACC CCGGGGTTAG CACCGTGGTG CTGGGTGCCT ATGACCTGAG GCGGCGGGAG AGGCAGTCCC GCCAGACGTT TTCCATCAGCAGCATGAGCG AGAATGGCTA CGACCCCCAG CAGAACCTGA ACGACCTGAT GCTGCTTCAG CTGGACCGTG AGGCCAACCT CACCAGCAGC GTGACGATAC TGCCACTGCC
  • the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence set forth in SEQ ID NOS: 1 , 3, 5, 7, 9, or 11.
  • the degree of identity between two or more amino acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48:443-453). For purposes of determining the degree of identity between two amino acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
  • amino acid sequences of the homologous polypeptides differ from the amino acid sequence set forth in SEQ ID NOS:l, 3, 5, 7, 9, or 11 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues.
  • amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the group of basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine) and small amino acids (such as glycine, alanine, serine, threonine and methionine).
  • Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, e.g., by H. Neurath and R.L.
  • the heparin binding protein may be encoded by a nucleic acid sequence having at least about an 80% identity with the nucleic acid sequence set forth in SEQ ID ⁇ O:2, which encodes human HBP having the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:4 which encodes human HBP having the amino acid sequence set forth in SEQ ID NO:3, or SEQ ID NO:6 which encodes human HBP having the amino acid sequence set forth in SEQ ID NO:5; or alternatively, SEQ ID NO: 8, which encodes porcine HBP having the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO: 10 which encodes porcine HBP having the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO: 12 which encodes porcine HBP having the amino acid sequence set forth in SEQ ID NO:l 1, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%, as determined by agarose gel electrophoresis.
  • the nucleic acid sequence may be of genomic, DNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • the degree of identity between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48:443-453).
  • GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.
  • Modification of the nucleic acid sequence encoding the HBP may be necessary for the synthesis of polypeptide sequences substantially similar to the HBP.
  • the term Asubstantially similar to the HBP refers to non-naturally occurring forms of the HBP.
  • These polypeptide sequences may differ in some engineered way from the HBP isolated from its native source. For example, it may be of interest to synthesize variants of the HBP where the variants differ in specific activity, thermostability, pH optimum, or the like using, e.g., site-directed mutagenesis.
  • the analogous sequence may be constructed on the basis of the nucleic acid sequence presented as the HBP encoding part of SEQ ID NOS:2, 4, 6, 8, 10, or 12, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the HBP encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence.
  • nucleotide substitution see, e.g., Ford et al., 1991, in Protein Expression and Purification 2:95-107.
  • amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for HBP activity to identify amino acid residues that are critical to the activity of the molecule.
  • a nucleic acid sequence encoding HBP may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3, 1984, pp. 801-805.
  • oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • the techniques used to isolate or clone a nucleic acid sequence encoding the heparin binding protein used in the method of the present invention are known in the art and include isolation from genomic DNA, preparation from DNA, or a combination thereof.
  • the cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g. , by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York.
  • nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligated activated transcription
  • NASBA nucleic acid sequence-based amplification
  • the nucleic acid sequence is then inserted into a recombinant expression vector which may be any vector which may conveniently be subjected to recombinant DNA procedures.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the nucleic acid sequence encoding HBP should be operably connected to a suitable promoter sequence.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the nucleic acid sequence encoding HBP in mammalian cells are the SV 40 promoter (Subramani et al., 1981, Mol. Cell Biol.
  • MT-1 metalothionein gene
  • adenovirus 2 major late promoter adenovirus 2 major late promoter
  • RSV Rous sarcoma virus
  • BPV bovine papilloma virus promoter
  • CMV cytomegalovirus
  • the nucleic acid sequences encoding SEQ ID NOS:2 and 9, e.g., SEQ ID NOS:4 and 10 may be operably linked to a nucleic acid encoding a heterologous pro sequence.
  • the nucleic acid encoding SEQ ID NOS:3 and 11, e.g., SEQ ID NOS:6 and 12 may be operably linked to a nucleic acid sequence encoding a heterologous signal sequence and/or pro sequence.
  • the nucleic acid sequence encoding HBP may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.)
  • the vector may further comprise elements such as polyadenylation signals (e.g., from SV 40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g., the SV 40 enhancer) and translational enhancer sequences (e.g., the ones encoding adenovirus VA RNAs).
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence (when the host cell is a mammalian cell) is the SV 40 or polyoma origin of replication.
  • the vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g., neomycin, ampicillin, geneticin or hygromycin.
  • a selectable marker e.g., a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g., neomycin, ampicillin, geneticin or hygromycin.
  • selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
  • the host cell may be a eucaryotic cell expressing an acidic proteoglycan(s) or mammalian hybrid cell.
  • the eucaryotic cell expressing an acidic proteoglycan(s) may be a basophilic cell or mast cell.
  • the mammalian basophilic cell may human, guinea pig, rabbit or rat basophilic cells
  • the mammalian basophilic cell is a rat basophilic cell.
  • the rat basophilic cell may be an RBL-1 cell having the identifying characteristics of ATCC CRL-1378 or RBL-2H3 cell having the identifying characteristics of
  • the mammalian basophilic cell is transfected with DNA encoding HBP using an electroporation apparatus.
  • the medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing mammalian cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection). The cells are then screened for antibiotic resistance.
  • the selected clones are subsequently assayed for HBP activity using assays known in the art such as a chemotaxis assay and assaying for cytokine release from monocytes (see, for example, Rasmussen et al, 1996, FEBS Lett. 390:109-112).
  • the host cell may be a hybrid mammalian cell.
  • a myeloma line e.g. , mouse, rat, human
  • a myeloma line e.g. , mouse, rat, human
  • a mammalian cell expressing an acidic proteoglycan such as a mammalian basophilic cell or mast cell using the following procedures.
  • the parental cells are mixed in culture media such as RPMI- 1640 and exposed to a chemical fusion agent such as polyethylene glycol (see, for example, Gefter et al, 1997, Somat. Cell Genet. 3:231-236).
  • the fusion agent is subsequently diluted out and the cells are incubated in media and HAT. Selected clones are subsequently assayed for HBP activity as described above.
  • two parental cells may be fused by electrofusion.
  • Membrane contact between cells are achieved by a non-uniform alternating field that leads to dielectrophoresis and cell chain formation. Fusion is then triggered by the injection of a field pulse that is strong enough to induce reversible breakdown in the membrane contact zone (see, for example, Okada et al., 1984, Biomed. Res. 5:511-566).
  • cell fusion may be induced by Sendai virus (see, for example, Wainberg et al., 1973, J. Cell Biol. 57:388-396).
  • the HBP produced by the recombinant host cells of the present invention may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g., ammonium sulphate, purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography, or the like.
  • the recombinant host cells may also produce an acid proteoglycan such as heparin sulfate. To obtain active HBP, the acid proteoglycan will need to be removed.
  • This may be accomplished using a series of separation methods, ie., precipitation or column chromatography, such as reverse phase HPLC, HIC, SEC, IEC and affinity based techniques.
  • the separation method may be combined with other treatments like increasing salt concentration, by change in the pH and by other means that reduce interactions between the acidic proteoglycan and HBP.
  • the vectors pBlueBacIII and pcDNA3 are obtained from Invitrogen. All primers and oligos are synthesized on an Applied Biosystems Model 394 DNA synthesizer.
  • RBL-1 cells (ATCC CRL-1378) and RBL-2H3 cells (ATCC CRL-2256) are obtained from American Type Culture Collection (ATCC) in Rockville, MD. Cells are grown as recommended by the supplier or in RPMI 1640 culture medium (Gibco, Life Technologies) supplemented with 10% heat inactivated gamma- irradiated FCS (origin: NZ, Gibco, Life Technologies) or fetal calf serum (FCS) North American origin from HyClone or BioWhittaker. Cells are grown in 5% CO 2 at 37BC in an 80% humidified atmosphere. Exponentially growing cells are used in all experiments.
  • FCS fetal calf serum
  • a 770 bp BamHl-Hindlll fragment is constructed using PCR technology from a human bone marrow DNA library (Clontech) based on the human HBP amino acid sequence (Flodgaard et al., 1991, Eur. J. Biochem. 197:535-547) and the CAP 37/azurocidin DNA sequence (Morgan et al., 1991, J. Immunol. 147:3210-3214 and Almeida et al., 1991, Biochem. Biophys. Res. Commun. 177:688-695).
  • This fragment contains the entire coding region of HBP, including a 19-residue signal peptide, a 7 amino acid pro-peptide, a mature part of 22 amino acids, and a 3 amino acid C-terminal extension.
  • the fragment is inserted into pBlue-BacIII resulting in the plasmid pSX556.
  • an oligonucleotide linker of 99 bp, covering the signal peptide and the first 4 amino acids of mature HBP is substituted for the original BamRl-Eagl fragment in pSX556 giving rise to pSX559.
  • pSX556 and pSX559 described above are used as templates in PCR reactions using the primers PBRa 246 (5'-CCGGGGATCCAACTAGGCTGGCCCCGGTCCCGG-3') (SEQ ID NO: 13)
  • PBRa247 (5'-CCGGGGATCCGATGACCCGGCTGACAGTCCTGG-3') (SEQ ID NO: 14) with a Pfu polymerase according to manufacturer's instructions (Stratagene).
  • Transfection is performed according to the following procedures. 25 ug of pcDNA3- HBP or pcDNA3-HBP ⁇ pro is transfected into RBL-1 cells or RBL-2H3 cells (8 x 10 6 cells are transfected using a BioRad Electroporation Apparatus with electric settings 960 uF and 300V as described by Gullberg et al., 1994, J. Biol. Chem. 269:25219-25225 and Garwicz et al., 1995, J. Biol. Chem. 270:28413-28418, or are transfected using LipofecAmine (Gibco, Life Technologies) or Superfect (Qiagen) transfection reagents as recommended by the suppliers.
  • LipofecAmine Gibco, Life Technologies
  • Superfect Qiagen
  • Cells are grown in RPMI 1640 medium supplemented with 10% heat inactivated gamma-irradiated FCS in 5% CO 2 at 37EC in an 80% humidified atmosphere. Geneticin (2 mg/ml) is added 48 hrs. post-transfection to select for recombinant clones.
  • HBP ELISA is a sandwich immunoassay using a monoclonal antibody as catcher and a polyclonal rabbit antibody conjugated to horseradish peroxidase as detector.
  • Antibodies are prepared according to standard procedures by immunizing mice and rabbits with HBP purified from human buffy coat eels (Flodgaard et al., 1991, Eur. J.
  • each well is coated with 0.5 :g monoclonal anti-hHBP dissolved in 100 :1 of PBS overnight.
  • the coated wells are washed three times with a solution of 5% lactose, 0.5% Byco A 0.05% Tween 20 and 0.024% thiomersal. After the last washing, the plates are left to dry at room temperature upside-down on a piece of cloth.
  • the coated plates are rapped with staniol and can be stored up to three months. Purified hHBP is used as reference preparation.
  • a working dilution of 100 ng hHBP/ml is prepared in a BSA-EDTA buffer and stored in aliquots at -80°C for a maximum of two weeks.
  • Serial dilution s containing 0; 0.3; 1 ; 4 and 12 ng hHBP/ml diluted in BSA- EDTA are made fresh and 100 :1 are added to each well.
  • hHBP samples are also diluted in BSA-EDTA buffer and all the samples are in-cubated agitated for 1 hour at room temperature.
  • the wells are emptied and washed three times with phosphate buffered saline followed by the addition of 100 :l/well diluted (1 : 1000) Fab-peroxidase conjugated rab-bit anti-hHBP, and incubated agitated for 1 hour at room temperature.
  • Peroxidase activity is measured using 100 :l/well TMB-perborate substrate solution, resulting in a color formation measurable photometrically at 450 nm.
  • the reference curve is linear when the logarithm to the absorbance is plotted against the logarithm to the dose.
  • Clones with the most pronounced expression are chosen for further experiments, recloned and retested for expression levels.
  • the highest HBP producers are selected and grown into mass culture or adapted to serum free or protein free medium.
  • the isolation of HBP from RBL-1 cells is carried out essentially as described by Rasmussen et al., 1996, FEBS Lett. 390:109-112.
  • the transfected and selected RBL-1 cells are initially filtered to remove any remaining cells and cell debris.
  • the culture medium is subsequently applied to a CM-Sepharose cation-exchange column (Pharmacia and Upjohn), previously equilibrated with 50 mM sodium phosphate, pH 7.3. Unbound and loosely bound materials are eluted with equilibration buffer until baseline is achieved measured by on-line UV detection at 280 nm.
  • the coulumn is then developed with a linear gradient from 0 to 1 M sodium chloride in equilibration buffer.
  • HBP eluted with about 0.7 M sodium chloride and fractions are combined based on UV absorption. Pooled fractions are diluted with two volumes of distilled water and applied on a new CM-Sepharose column. Following equilibration HBP is step-eluted with 1 M sodium chloride in equilibration buffer and fractions combined based on absorption at 280 nm. Highly concentrated and pure HBP is obtained by this procedure. Final purification is carried out on a Sephadex G-25 gel-filtration column (Pharmacia & Upjohn) equipped with a UV-flow cell and equilibrated and eluted with 0.02% trifluoroacetic acid. HBP is collected based on absorption at 280 nm. The gel filtration serves mainly as a buffer exchange step to produce a stable preparation of HBP that is kept at 4BC until use.
  • RBL-1 HBP Recombinant HBP obtained from RBL-1 cells
  • RBL-1 HBP is analyzed by SDS-gel electrophoresis, peptide and carbohydrate mapping, mass spectrometry (MS), capillary electrophoresis (CE), and HPLC and compared to recombinant HBP obtained from insect cells (Rasmussen et al., 1996, FEBS Lett. 390:109-112), hereinafter referred to as insect cell HBP, and native HBP obtained from polymorphonuclear leukocytes (Flodgaard et al., 1991, Eur. J. Biochem. 197:535-547), hereinafter referred to as human HBP.
  • insect cell HBP insect cell HBP
  • human HBP native HBP obtained from polymorphonuclear leukocytes
  • RBL-1 HBP and insect cell HBP is tested on SDS-PAGE (see Figure 2). It appears that RBL-1 HBP has a molecular wieght slightly larger than insect cell HBP.
  • HBP and RBL-1 HBP are homogenous with regard to their N-terminal but heterogenous with regard to their C-terminal.
  • RBL-1 HBP contained the three peptide forms HBP 1-221, HBP 1-223 and HBP 1-225.
  • Human HBP contains the two peptide forms HBP 1-221 and HBP 1-223, and possibly also the HBP 1-225 peptide.
  • HBP from baculovirus infected insect cell culture (insect cell HBP) is used as reference. Insect cell HBP contains two peptide forms: HBP 1-225 peptide (80-90%) and HBP 5-225 peptide (10- 20%, tHBP).
  • Samples of human, RBL-1 and insect cell HBP, respectively, are desalted into a 10 mM Mes buffer, pH 5.9 on NAPS columns. A volume containing about 50 p9 HBP is withdrawn each sample and concentrated to 10 FI in a Speedvac concentrator. Mes and CaC12 buffers, pH 5.9 are added to a final concentration of 100 mM Mes, 10 mM CaCl 2 in 30 pi. Following addition of 20 FI 7 M ion-exchanged urea, the samples are allowed to denature for 10 min. before injection on a Poroszyme immobilized trypsin cartridge (Perceptive Biosystems, part nr.
  • each collected digest (about 8 Fg HBP before digestion) are analysed on a RP-HPLC Vydac 218 TP52 (250 mm x 2.1 mm i.d.) column using an Applied Biosystemsl30 HPLC system and a linear gradient of 3-80 % B (0.055 % TFA, 70 % acetonitrile) over 65 min.
  • the tryptic peptide maps of human -, RBL-1- and insect cell HBP, respectively, obtained following digestion on a Poroszyme immobilised trypsin cartridge and separation on a Vydac Cl 8 RP-HPLC column are shown in Figure 3. It is seen that the peptide maps of human HBP and RBL-1 HBP are very similar and that they differ considerably from the peptide map of insect cell HBP.
  • the monosaccharide composition is determined by AIE-HPLC with pulsed amperometric detection.
  • the results are consistent with the results from both carbohydrate map and MALDI-MS (see below), is that the dominating structures are a fucosylated core-structure (Fuc-GlcNAc2-Man3) and truncated forms thereof. Larger complex structures are indicated in low amounts for human HBP by the galactose content and in moderate amounts for RBL-1 HBP by the galactose and N-acetylgalactosamine contents.
  • the carbohydrates are released by hydrazinolysis on a GlycoPreplOOO unit using N+O mode (434-084) and labelled with 2-aminobenzemide (2-AB) using a labelling kit (K-404, Oxford GlycoSystems).
  • the 2-AB carbohydrate pools are analysed by IE-HPLC ( Figure 4) on a CarboPac PA- 100 column eluted with a NaAc gradient in NaOH.
  • the pools are desialylated by sialidase treatment (Sialidase X-5022, Oxford GlycoSystems, 37EC, 16 hrs) and analysed by IE-HPLC ( Figure 4) and size exclusion chromatography (Figure 6) on a Glycan Sizing column eluted with water and calibrated in glucose units (g.u.).
  • fluorescence detection is used (exe. 330 nm, emm. 420 nm).
  • Core-structures Structure A: Truncated core-structure or non-carbohydrate impurity.
  • Structure B Core-structure with fucose, lacking one mannose, 4.8 g.u., 876.3 Da.
  • Structure C Core-structure with fucose, 5.8 g.u., 1038.4 Da.
  • Structure D Core-structure without fucose, 4.8 g.u., 892.3 Da.
  • the core-structures represent approx. 80%) of the human HBP carbohydrates, approx. 50% of the RBL-1 HBP carbohydrates, and 100% of the insect cell HBP carbohydrates.
  • Structures E-F and neighbouring structures eluted as mono- and possibly disialylated structures (IE-HPLC, Figure 4).
  • the carbohydrates eluted as neutral mono- or biantenna complex structures, ie. structures from 9 to 13 g.u. (IE-HPLC, Figure 5, SEC, Figure 6).
  • the sialylated structures represent approx. 20% of the human HBP carbohydrates and approx. 40% of the RBL-1 HBP carbohydrates.
  • the late-eluting structures ( Figure 4) eluted as highly sialylated structures, but the retention times were un-changed after sialidase treatment (not apparent in Figure 5 due to change in the gradient compared to Figure 4).
  • the late-eluting structures represented approx. 10% of the RBL- 1 HBP carbohydrates.
  • MALDI-MS is performed on the three proteins using sinapinic acid as matrix.
  • the main mass found by MALDI-MS of insect cell HBP ( Figure 5) is 27236 Da.
  • RBL-1 HBP ( Figure 7) the main mass of 26715.6 Da can be interpreted as the HBP 1 -223 peptide with three core-structures with fucose and lacking one mannose (structure B), which has a theoretical mass of 26722 Da.
  • the mass spectrum shows a large amount of structures in the range 27-30 kDa, which can be assigned to glycoforms with one or more of the carbohydrate structure(s) B substituted with eg. sialylated structure(s); each substitution would add 600-1500 Da depending on the structure added.
  • the main mass of 26681.9 Da can be the same structure, i.e., the HBP 1-223 peptide with three structures B.
  • CE Capillary Electrophoresis
  • PVA polyvinylalcohol
  • the other system is a neutral buffer, 25 mM phosphoric acid with 0.005% poly- Arginine, titrated to pH 6.5 with ammonium hydroxide.
  • Poly-Arginine adsorbs to the fused silica wall through strong ionic interaction. The net charge of the wall becomes positive.
  • Analyses of capture eluate of insect cell HBP indicate that the two above-mentioned buffer systems have different selectivities. Therefore, the two test substances, RBL-1 HBP and human HBP, are analyzed in both buffer systems. Before analysis they are transferred to 10 mM MES pH 5.9 by gel filtration (NAP 5 column). Capture eluate of insect cell HBP is used as a reference.
  • Figures 8 and 9 show the separations of the three HBP samples in the two above mentioned buffer systems. Clearly, the figures show that the two buffers have different selectivities and that the microheterogeneity of the samples is highly different. However, there are also some similarities, which may be interpreted, takinn into consideration the data from other analyses.
  • human HBP and RBL-1 HBP have a series of peaks eluting after the predominant peaks.
  • the neutral poly-Arg buffer Figure 9) correspondingly peak complexes eluting prior to the main peak are observed. The intensity of the peaks is largest in RBL-1 HBP. At the same time insect cell HBP has virtually no peaks at the same positions.
  • CIE-HPLC CIE-HPLC is performed on a MonoS HR 5/5 column eluted with a NaCl gradient in 20 mM Ches, pH 9.0 with UV detection at 280 nm. Samples are desalted to 10 mM MES, pH 5.9, before analysis. Human HBP elutes as two peaks, RBL-1 HBP eluted as three peaks and insect cell HBP elutes as one main peak with a front peak representing tHBP ( Figure 10). Although all the main-peaks for the three HBP forms are closely-eluting, none of them is co- eluting ( Figure 10). These results may be due to the C-terminal heterogeneity.
  • GP-HPLC is performed on a Waters Protein-Pak 125, 7.8 mm x 300 mm, eluted with 0.2 M ammonium sulfate, 5% isopropanol, pH to 2.5 with phosphoric acid, then to pH 5.0 with triethylamine. UV detection at 215 nm. Samples are desalted to 10 mM MES, pH 5.9, before analysis.
  • the GP-HPLC profiles of human HBP and of RBL-1 HBP show one broad peak (Figure 11). Human HBP elutes in a slightly higher elution volume compared to insectcell HBP and with a significant shoulder on the front (Figure 11). RBL-1 HBP elutes in the same elution volume as insect HBP but with a very large shoulder on the front ( Figure 11). These results could be due to the glycoform heterogeneity proposed from the carbohydrate mapping and MALDI-MS.
  • GTCTTCAGCC GCCGGGGCCG CATCAGCCAG GGAGACAGAG GCACCCCCCT CGTCTGCAAC 540 GGCCTGGCGC AGGGCGTGGC CTCCTTCCTC CGGAGGCGTT TCCGCAGGAG CTCCGGCTTC 600
  • TGTCGCCCCA ACAACGTGTG CACCGGTGTG CTCACCCGCC GCGGTGGCAT CTGCAATGGG 540 GACGGGGGCA CCCCCCTCGT CTGCGAGGGC CTGGCCCACG GCGTGGCCTC CTTTTCCCTG 600
  • GCCACGGTGG AAGCCGGCAC CAGATGCCAG GTGGCCGGCT GGGGGAGCCA GCGCAGTGGG 480 GGGCGTCTCT CCCGTTTTCC CAGGTTCGTC AACGTGACTG TGACCCCCGA GGACCAGTGT 540
  • CTTTTCTCCC GCTTCCCAAG GGTGCTCAAT GTCACCGTGA CCTCAAACCC GTGTCTCCCC 540

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur un procédé d'obtention de protéines fixant l'héparine dans des cellules de mammifères de recombinaison, et en particulier dans des cellules eucariotes produisant du protéoglycan, et dans des cellules hybrides de mammifères. L'invention porte également sur lesdites cellules de mammifères de recombinaison, et sur leurs méthodes d'obtention. L'invention est illustrée par les cellules basophiles RBL-1 de la leucémie du rat.
PCT/DK1998/000275 1997-06-25 1998-06-22 Procede d'obtention de proteines fixant l'heparine dans des cellules de mammiferes Ceased WO1999000417A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU80124/98A AU8012498A (en) 1997-06-25 1998-06-22 Production of heparin-binding protein in mammalian cells

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US8241397P 1997-06-25 1997-06-25
US60/082,413 1997-06-25
DK0773/97 1997-06-30
DK77397 1997-06-30
US5519297P 1997-08-11 1997-08-11
US60/055,192 1997-08-11
DK0963/97 1997-08-22
DK96397 1997-08-22

Publications (1)

Publication Number Publication Date
WO1999000417A1 true WO1999000417A1 (fr) 1999-01-07

Family

ID=27439374

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1998/000275 Ceased WO1999000417A1 (fr) 1997-06-25 1998-06-22 Procede d'obtention de proteines fixant l'heparine dans des cellules de mammiferes

Country Status (1)

Country Link
WO (1) WO1999000417A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066627A1 (fr) * 1999-04-29 2000-11-09 Leukotech A/S Expression de la proteine de fixation d'heparine dans des cellules mammiferes de recombinaison
US6417173B1 (en) 1991-05-31 2002-07-09 Gliatech, Inc. Methods and compositions based on inhibition of cell invasion and fibrosis by anionic polymers
WO2003080660A3 (fr) * 2002-03-27 2003-12-18 Leukotech As Procede de preparation d'une proteine de liaison a l'heparine (hbp) mammifere recombinee

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028949A1 (fr) * 1994-04-21 1995-11-02 Novo Nordisk A/S Proteine de liaison de l'heparine destinee au traitement de la septicemie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028949A1 (fr) * 1994-04-21 1995-11-02 Novo Nordisk A/S Proteine de liaison de l'heparine destinee au traitement de la septicemie

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 270, No. 21, May 1995, URBAN GULLBERG, "Carboxyl-Terminal Prodomain-Deleted Human Leukocyte Elastase and Cathepsin G are Efficiently Targeted to Granules and Enzymatically Activated in the Rat Basophilic/Mast Cell Line RBL", pages 12912-12918. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 270, No. 47, November 1995, DANIEL GARWICZ et al., "Human Cathepsin G Lacking Functional Glycosylation Site is Proteolytically Processed and Targeted for Storage in Granules After Transfection to the Rat Basophilic/Mast Cell Line RBL or the Murine Myeloid Cell....", pages *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6417173B1 (en) 1991-05-31 2002-07-09 Gliatech, Inc. Methods and compositions based on inhibition of cell invasion and fibrosis by anionic polymers
US6756362B2 (en) 1991-05-31 2004-06-29 Dikla Roufa Methods and compositions based on inhibition of cell invasion and fibrosis by anionic polymers
WO2000066627A1 (fr) * 1999-04-29 2000-11-09 Leukotech A/S Expression de la proteine de fixation d'heparine dans des cellules mammiferes de recombinaison
WO2003080660A3 (fr) * 2002-03-27 2003-12-18 Leukotech As Procede de preparation d'une proteine de liaison a l'heparine (hbp) mammifere recombinee

Similar Documents

Publication Publication Date Title
Wisniewski et al. TSG-6, an Arthritis-Associated Hyaluronan Binding Protein, Forms a Stable Complex with the Serum Protein Inter-. alpha.-inhibitor
Spik et al. A novel secreted cyclophilin-like protein (SCYLP)
Letellier et al. Mechanisms of formation of IgE-binding factors (soluble CD23)—I. Fcϵ R II bearing B cells generate IgE-binding factors of different molecular weights
US5312736A (en) Anticoagulant analogues of the tissue factor extrinsic pathway inhibitor (EPI) with reduced affinity for heparin
EP1346034B1 (fr) Polypeptide de la protease de clivage du facteur de von willebrand (vwf), acide nucleique codant ce polypeptide et utilisation de ce polypeptide
Yurchenco et al. Recombinant laminin G domain mediates myoblast adhesion and heparin binding.
Jin et al. Separation, purification, and sequence identification of TGF-β1 and TGF-β2 from bovine milk
JP4230659B2 (ja) ケモカインアンタゴニストとしてのアミノ末端切除型ｍｃｐ−２
VOSS et al. Expression of human interferon ω1 in Sf9 cells: No evidence for complex‐type N‐linked glycosylation or sialylation
CA2329054A1 (fr) Nouveau polypeptide phosphatonine hormonale
Samy et al. Primary structure of macromomycin, an antitumor antibiotic protein.
MX2012002129A (es) Purificacion del factor de von willebrand (vwf) para remocion incrementada de virus envueltos sin lipido.
Choi-Miura et al. Purification and characterization of a novel glycoprotein which has significant homology to heavy chains of inter-α-trypsin inhibitor family from human plasma
Scoble et al. [28] Characterization of recombinant proteins
WO1999000417A1 (fr) Procede d'obtention de proteines fixant l'heparine dans des cellules de mammiferes
Gasnier et al. Characterization and location of post‐translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules
Rhodes et al. Isolation and characterization of the cyanogen bromide peptides derived from the human. alpha. 2 (V) collagen chain
JP3637087B2 (ja) 新規な白血球活性化因子
KR20020034073A (ko) 재조합 포유동물 세포에서 헤파린-결합 단백질의 발현
WO1999000416A1 (fr) Utilisation de proteines fixant l'heparine pour empecher la penetration d'elements pathogenes dans des cellules mononucleaires
US7547676B2 (en) Antagonist peptides to the C5A chemotactic function of vitamin D binding protein
CA2042399A1 (fr) Isolement d'un polypeptide physiologiquement actif a partir de la thrombomoduline humaine
Pedersen et al. Octapeptide segments from the amino terminus of glycophorin A contain the antigenic determinants of the M and N blood groups systems
Zhao et al. Purification and characterization of a 95-kDa protein—carcinoembryonic antigen-related cell adhesion molecule 8—from normal human granulocytes
CA2340790A1 (fr) Adn humain il-1 epsilon et polypeptides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1999505224

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA