KR20020034073A - Expression of heparin-binding protein in Recombinant mammalian cells - Google Patents

Abstract

The present invention comprises the steps of (a) introducing a nucleic acid encoding a mammalian heparin-binding protein (HBP) into a mammalian cell; (b) culturing the cells of step (a) under conditions inducing expression of HBP; And (c) recovering the HBP in a culture medium, wherein the nucleic acid encoding the heparin-binding protein is introduced into the cell, and then the method produces the heparin-binding protein in a mammalian cell that can be cultured under anaerobic conditions. It is about. In addition, the present invention used in this method relates to the recombinant mammalian cells described above.

Description

Expression of heparin-binding protein in Recombinant mammalian cells

The covalent structure of two closely related proteins isolated from peripheral neutrophil leukocytes derived from human and swine has been recently discovered (cf. H. Flodgaard et al., 1991, Eur. J. Biochem. 1997: 535-547; J. Pohl et al., 1990, FEBS Lett. 272: 200 ff.). Both proteins show high similarity to neutrophil elastase, but selective mutations of active serine 195 and histidine 57 (chimodlipsine numbering (BS Hartley, 1970, Phil. Trans. Roy. Soc. Series 257: 77 ff.) These proteins lack proteolytic activity because they show high affinity for heparin, so they are named human heparin-binding protein (hHBP) and porcine heparin-binding protein (pHBP), respectively; Has named this protein cationic antimicrobial protein (CAP37) because of its antimicrobial activity (WM, Schafer et al., 1986, Infect. Immun. 53: 651 ff.) This protein is known as chemotaxis, It has been found to regulate the function of monocytes / macrophages, such as increased viability, differentiation, etc. (Pereira, 1995, J. Leuk. Biol. 57: 805-812, see also US Pat. Nos. 5,458,874 and No. 5,484,885). Or it not has been disclosed about how to infection by microbes are inhibited monocyte or macrophage.

Moreover, when HBP was treated to endothelial cells and fibroblasts cultured in monolayer culture, HBP was shown to mediate the datechment and contraction of endothelial cells and fibroblasts. HBP also stimulates the survival of monocytes and the secretion of thrombospondin secretion (E, Ostergaard and H. Flodgaard, 1992, J. Leukocyte Biol. 51: 316 ff).

In addition, proteins with the first 20 N-terminal amino acid residues identical to the amino acid sequence of hHBP and CAP37, called azurocidin, were isolated from azurophil granules (J, E. Gabay et al. , 1989, Proc. Nat I. Acad. Sci. USA 86: 5610 ff .; CG Wilde et al., 1990, J. Biol. Chem. 265: 2038 ff.), Their antimicrobial properties have been reported (D. Campaneili). et al., 1990, J. Clin. Invest 85: 904 ff.).

The presence of hHBP in neutrophil leukocytes and the fact that 89% of CAP37 (same as hHBP) is released when leukocytes phagocytose Staph. Aureus (HA Pereira et al, cited above) .) Indicates that the function of hHBP may be involved in the inflammatory process since it is clear that the protein is released from neutrophils. Since Pereira et al. Cited above have the ability to attract monocytes at the site of inflammation, CAP37 is one of the factors leading to the influx of monocytes in the second wave of inflammation. Suggested. Ostergaard and Flogadard cited above indicate that neutrophils as well as extravasation of monocytes, in addition to HBP, play an important role in attracting monocytes (to the target site). It also suggested that it plays an important role.

The structure of HBP is disclosed in US Pat. No. 5,814,602 and in H. Floydgaard et al., Cited above. HBP is described in CAP37 (cf. WO 91/00907, US Pat. Nos. 5,458,8745 and 5,484,885) and Azurdine (cf. CG Wilde et al., J. Biol. Chem. 265,1990, p. 2038) It is also named otherwise.

HBP was previously isolated from neutrophil leukocytes (Flodgaard et al., 1991, Eur. J. Biochem. 197: 535-547). However, the yield was very low. Moreover, HBP was also produced by recombinant DNA methods in insect cells (Rasmussen et al., 1996, FEBS Lett. 390: 109-112).

Recombinant HBP (referred to as CAP37 in the literature) was also prepared from human kidney 293 cell line (R. Alberdi et al., 1997, FASEB J. 11: 1915). RSV-PL4 expression vector was used. This vector contains a secreted transferrin signal peptide, an HPC4 epitope for immunocompatible purification, a factor Xa cleavage site and a neomycin resistance gene for G418 selection. Functional heparin-binding proteins were prepared by in vitro cutting the Xa factor cleavage site of the recombinant protein with bovine Xa factor to separate the fusion peptide from the recombinant heparin-binding protein.

In addition, recombinant HBP was produced in reticulocyte blood leukocytes (RBLs) (PCT / DK98 / 00275). However, heterogeneous populations of HBP with varying glycosylation are obtained. It is useful to produce heparin-binding proteins in high yield by simple but efficient methods. Accordingly, it is an object of the present invention to obtain mature heparin-binding proteins in high yield by a simple but efficient method.

Summary of the Invention

The present invention comprises the steps of (a) introducing a nucleic acid encoding a heparin-binding protein into mammalian host cell (s) that can be cultured under anaerobic conditions after introduction of the nucleic acid; (b) culturing the cell (s) of step (a) under conditions inducing expression of the HBP; And (c) recovering the HBP from the culture medium, wherein the mammalian heparin-binding protein can be cultured under anaerobic conditions after introducing the nucleic acid encoding the heparin-binding protein into the cell (s). It is about how to produce. In certain embodiments, the cells of step (b) are cultured in the presence of a bradykinin B-2 receptor antagonist.

"Heparin binding protein" ("HBP") is (i) proteolytic activity is inactive; (ii) stored in Jourgerian granules of polymorphonuclear leukocytes; (iii) a protein that is a chemoattractant for monocytes, and optionally a protein having a glycosylated form of about 27-31 kD molecular weight. HBP is derived from human or swine.

In another embodiment, the present invention provides a method for producing a nucleic acid encoding a heparin-binding protein in mammalian host cell (s) that can be cultured under anaerobic conditions after introduction of the nucleic acid; (b) selecting a mammalian host cell of step (a) comprising a nucleic acid encoding a heparin-binding protein; (c) culturing the host cell in a serum-free medium and optionally a medium containing no protein; And (d) isolating said heparin-binding protein. The method may further comprise purifying the HBP described above.

The invention also relates to a recombinant mammalian host cell which can be cultured under anaerobic conditions after introduction of a nucleic acid encoding a mammalian heparin-binding protein and comprises a nucleic acid sequence encoding a heparin-binding protein.

The present invention relates to a method for producing a Heparin-binding protein (HBP) in a recombinant mammalian cell (s), wherein the recombinant mammalian cell (s) is a nucleic acid encoding the heparin-binding protein. Cell (s) that can be cultured under anaerobic conditions after introduction into the cell. The invention also relates to the recombinant mammalian cells described above.

Heparin binding proteins are determined by agarose gel electrophoresis, as shown in SEQ ID NO: 3 (SEQ ID NO: 1 encoding the mature human HBP), SEQ ID NO: 5 (SEQ ID NO: 6, signal sequence and mature protein). A sequence encoding a human HBP comprising a sequence), SEQ ID NO: 7 (SEQ ID NO: 8) and a sequence encoding a human HBP comprising a signal sequence, a pro sequence, and a sequence of mature protein) or SEQ ID NO: 4 (SEQ ID NO: A sequence encoding a porcine HBP represented by 2), SEQ ID NO: 9 (SEQ ID NO: 10, a sequence encoding a porcine HBP including a signal sequence and a sequence of mature protein), SEQ ID NO: 11 (SEQ ID NO: 12) At least about 80%, preferably at least about 90%, and more preferably at least about 80% of the nucleic acid sequence described in the sequence encoding a porcine HBP comprising a signal sequence and a sequence of a mature protein). Degree of between about 95% and most preferably may be encoded by the nucleotide sequence having a homology of about 97% at least. The nucleic acid sequence may be genome, cDNA, RNA, semisynthetic, synthetic derived or any combination thereof.

The degree of homology between two nucleic acid sequences can be determined using 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). In the present invention, for the purpose of determining the degree of homology between two nucleic acid sequences, a GAP set as follows was used: a GAP product penalty of 5.0 and a GAP expansion penalty of 0.3.

Modification of nucleic acid sequences encoding HBP may be required for the synthesis of polypeptide sequences substantially similar to HBP. The term "substantially similar" for HBP refers to a form of HBP that does not occur naturally. The sequences of these polypeptides may differ from HBP isolated from their original origin in some engineered way. For example, it may be advantageous to synthesize variants of HBP that differ in specific activity, thermostability, optimal pH, etc., for example using site-directed mutagenesis. Such analogous sequences are based on nucleic acid sequences present as HBP coding moieties of SEQ ID NO: 1 or 2, eg, sub-sequences thereof; And / or by the introduction of nucleotide substitutions which occur not frequently in other amino acid sequences of the HBP encoded by the nucleic acid sequence but correspond to the codon use of the host organism intended for the production of the enzyme; Or by the introduction of nucleotide substitutions that can lead to other amino acid sequences. For a general description of nucleotide substituents, see Ford et al., 1991, in Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutions may result in polypeptide sequences that are still active when made outside the region critical to the function of the molecule. An amino acid residue region, which is essential for the activity of a polypeptide encoded by a nucleic acid sequence isolated in the present invention, and which preferably does not perform substitution, may be site-directed mutagenesis or alanine-scanning mutagenesis (e.g., For example, Cunningham and Wells, 1989, Science 244: 1081 1085) can be found according to methods known in the art. In the following technique, mutations are performed at every residue in the molecule, and HBP activity is examined for the resulting mutant molecules to determine amino acid residues that are critical to the activity of the molecule.

In addition, heparin-binding proteins are encoded by nucleic acid sequences capable of forming hybrids with the nucleic acid sequences set forth in SEQ ID NOs: 3, 4, 5, 7, 9 or 11 under low to high stringency conditions. Can be. High and low stringency conditions were 5 × SSPE, 0.3% SDS, 200 μg / ml digested, denatured salmon sperm DNA, and low, medium or high stringency conditions, 25, 35 or 50% form respectively. It is defined as prehybrid and hybrid formation at 42 ° C. in a solution containing amide. The carrier materials are each preferably at least 50 ° C. (very low stringency), more preferably at least 55 ° C. (low stringency), more preferably at least 60 ° C. (medium stringency), more preferably at least 65 ° C. (medium). High stringency), even more preferably wash three times for 30 minutes using 2X SSC, 0.2% SDS at least 70 ° C. (high stringency) and most preferably at least 75 ° C. (very high stringency).

Preparation of HBP

Nucleic acid sequences encoding HBP are established by standard methods such as S. L. Beaucage and M. H, Caruthers, Tetrahedron Letters 22,1981, pp. Phosphoamidite method described in 1859-1869 or Matthes et al., EMBO Journal 3,1984, pp. It can synthesize | combine and manufacture by the method of 801-805. According to the phosphoramidite method, oligonucleotides are synthesized, for example with an automated DNA synthesizer, isolated, annealed, ligated and cloned in a suitable vector.

Techniques used to isolate or clone nucleic acid sequences encoding heparin binding proteins used in the methods of the invention are known in the art and include isolation from genomic DNA, preparation from cDNA, or combinations thereof. Cloning of nucleic acid sequences according to the present invention from such genomic DNA can be performed, for example, by the use of known polymerase chain reaction (PCR) or expression libraries to detect cloned DNA fragments having shared structural characteristics. This can be accomplished using antibody screening (see, eg, Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York). Other nucleic acid amplification methods can be used, such as ligase chain reaction (LCR), ligated activated transcription (LAT), and nucleic acid sequence-based amplification (NASBA).

The nucleic acid sequence can then be inserted into any recombinant expression vector that can be conveniently used in recombinant DNA methods. The choice of vector will often depend on the host cell being introduced. Thus, the vector may be a vector that replicates autonomously, i.e., an entity other than a chromosome, and is a vector, eg, a plasmid, capable of replicating independently of chromosomal replication. In addition, such vectors, when introduced into a host cell, may be integrated into the host cell genome and replicated with the integrated chromosome.

In a vector, the nucleic acid sequence encoding the HBP must be operably linked to a suitable promoter sequence. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the selected host cell, and may be derived from a gene encoding a protein homologous or heterologous to the host cell. Examples of preferred promoters for eliciting transcription of nucleic acid sequences encoding HBP in mammalian cells include the SV40 promoter (Subramani et al., Mol. Cell Biol. 1, 1981, pp. 854-864), MT-1 (Metallothionein). gene) Palmiter et al., Science 222, 1983, pp. 809-814 or adenovirus 2 major late promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus ( cytomegalovirus (CMV) promoter (Boshart et al., 1981, Cell 41: 521-530) and bovine papilloma virus promoter (BPV). Suitable promoters for use in insect cells are polyhedrin promoters (Vasuvedan et al., FEBS Lett. 311,1992, pp. 7-11).

Nucleic acid sequences encoding HBP may also be operably linked to suitable transcription terminators, such as human growth hormone transcription terminators (Palmiter et al., Cited above).

The vector can also contain polyadenylation signals (eg, from the SV40 or aneovirus 5 Elb region), transcriptional enhancer sequences (eg, SV40 enhancers) and translation enhancer sequences (eg, adenovirus VA RNAs). May be further included).

The recombinant expression vector may further include a DNA sequence which allows the vector to be replicated if a problem occurs in the host cell. Examples of such sequences (when the host cell is a mammalian cell) include an SV40 or polyoma origin of replication. polyoma origin of replication).

The vector may also be a gene that encodes a gene product that complements the defects of the host cell, such as a gene encoding a selection marker, e.g., dihydrofolate reductase (DHFR), or neomycin, geneticin. And genes that confer resistance to drugs such as ampicillin or hygromycin.

In certain embodiments, the present invention relates to a method for producing HBP, wherein a host cell containing a DNA sequence encoding a mature HBP extended to an N-terminal dml forward site is subjected to expression for HBP. Cultivation is carried out using a suitable culture medium, and the resulting HBP is recovered from the culture medium to N-terminal expanded HBP.

The extension of the N-terminus may be a sequence consisting of about 5-25 amino acid residues, in particular about 8-15 amino acid residues. Amino acid residues in the N-terminal sequence are not believed to play a decisive role. N-terminal extension is peptide or prepropeptide Met-Thr-Arg-Leu-Thr-Val-Leu- of HBP having the amino acid sequence Gly-Ser-Ser-Pro-Leu-Asp (SEQ ID NO: 13) Ala-Leu-Leu-AlaGly-Leu-Leu-Ala-Ser-Ser-Arg-Ala-Gly-Ser-Ser-Pro-Leu-Leu-Asp (SEQ ID NO: 14) may be suitable.

To facilitate the production of mature HBP, the DNA sequence encoding the N-terminal extended HBP is positioned between the DNA sequence encoding the N-terminal extension and the DNA sequence encoding the mature HBP. It is generally preferred to include the DNA sequence encoding. Examples of preferred protease cleavage sites are enterokinase cleavage sites or Xa factor cleavage sites having the amino acid sequence of interest.

In addition, nucleic acid sequences encoding signal sequences and mature HBP can be inserted into the vector using the methods described above. As a result, HBP can be separated from the culture medium.

The method used to lignate the nucleic acid sequence, promoter and transcription terminator encoding HBP or N-terminal extension, respectively, and the method used to insert the ligated vector into a suitable vector containing the information needed for replication, Known to those skilled in the art (cf., eg, Sambrook et al., Cited above).

Mammalian cells are cells that can be cultured under anaerobic conditions after transfection with a nucleic acid encoding a mammalian heparin-binding protein. In a more preferred embodiment, the mammalian cell is an adenovirus-transformed cell (s) or cell (s) derived from an embryonic cell. As defined herein, cells derived from embryonic cells are cell (s) obtained from a primary culture of embryonic cells or cell (s) obtained from cell lines initially passaged from primary cultures of embryonic cells. Examples of adenovirus-transformed cells or cells derived from embryonic cells are human embryonic kidney (HEK) cells, in particular HEK 293 cells.

Methods for transfecting mammalian cells and for expressing DNA sequences introduced into cells are described, for example, in Kaufman and Sharp, 1982, J. Mol. Biol. 159: 601-621; Southern and Berg, 1982, J. Mol. Appl. Genet. 1: 327-341; Loyter et al., 1982, Proc. NatI. Acad. Sci. USA 79: 422-426; Wigler et al., 1978, Cell 14: 725; Corsaro and Pearson, 1981, Somatic Cell Genetics 7: 603, Graham and van der Eb, 1973, Virology 52: 456; Fraley et al., 1980, JBC 225: 10431; Capecchi, 1980, Cell 22: 479; Wiberg et al., 1983, NAR 11: 7287; and Neumann et al., 1982, EMBO J. 1: 841-845. Insect cells are preferably transfected with a baculovirus vector disclosed in US Pat. No. 4,745,051.

The medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as serum-containing or serum-free medium containing appropriate additives, and may be a suitable medium for growing insect cells. . Suitable media can be commercially available and can be prepared according to published methods (eg, as described in the catalog of the American Type Culture Collection). Next, the cells are screened for antibiotic resistance. Subsequently, the selected clones were tested for HBP activity using conventional methods known in the art, such as chemoaxis assays and cytokine release from monocytes (see, eg, US Pat. No. 5,814,602). do.

The selected clones can be further cultured in medium without serum and optionally no protein. It can also be cultured in the presence of a bradykinin B-2 receptor antagonist. However, such examples are not limited to the bradykinin B-2 receptor antibody (see, eg, Haasemann et al., 1991, J. Immunol. 147: 3882-3992) or antisense polynucleotide sequences for the bradykinin receptor gene. .

The HBP produced by the cells is separated from the host cell by medium by centrifugation or filtration; Precipitating the proteinaceous component of the supernatant or filtrate with a salt such as ammonium sulfate; It may be recovered from the culture medium by conventional methods including the purification by various chromatography methods such as ion exchange chromatography, affinity chromatography, and the like. In certain embodiments, the N-terminally expanded HBP is purified by chromatography of an agaroselinked aprotinin column.

If the HBP is HBP with N-terminus expanded, after recovery from the culture medium, the N-terminus expanded HBP can be cleaved with a suitable proteolytic enzyme to produce mature (and activated) HBP. Examples of suitable enzymes include, but are not particularly limited to, enterokinase and Xa factor.

Example 1: Expression of pro-HBP in insect cells

The method used to construct pSX556 is described in Rasmussen et al., 1996, FEBS Lett. 390: 109-112.

The following PCR primers were synthesized:

MHJ 2087: 5'-AAA AAG GAT CCA CCA TGA CCC GGC TGA CAG TCC TGG CCC TGC TGG CTG GTC TGC TGG CGT CCT CGA GGG CCG GCT CCA GCC CCC TTT TGG ACA TCG TTG GCG GCC GGA AGG C-3 '(SEQ ID NO: : 15)

MHJ 2089: 5'-AAA AAA GCT TCC TAG GCT GGC CCC GGT CCC GGA TTG TTT AAA ACG CCA TC-3 '(SEQ ID NO .: 16)

MHJ 2087 is initially derived from the Bam HI site, the start codon and the prepro portion of human cDNA (Morgan, JG et al., 1991, J. Immun., 147: 3210-3214) and the beginning of the linked gene mature portion. Encode 20 nucleotides.

MHJ 2089 is complementary to the coding portion of the HBP gene and the last eight codons from two external codons according to the cDNA cited above.

PCR was performed using the following two primers.

3 cycles: 60 seconds at 95 ° C, 120 seconds at 50 ° C, 120 seconds at 72 ° C

12 cycles 30 seconds at 95 ° C., 60 seconds at 65 ° C., 90 seconds at 72 ° C.

The PCR product 760bp fragment was isolated by electrophoresis on a 1% agarose gel, digested with Bam HI and Hind III and inserted into pSX221 digested with the same two enzymes (pSX221 is a derivative of pUC19 (Yannisch-Perron , C. et al., 1985, Gene 33: 103-119). Cloned DNA was confirmed by sequencing, Bam HI- Hind III fragments were cut and then isolated and inserted into pBlueBacIII (Invitrogen Corporation) for expression in insect cells. This fragment comprises the entire coding region of HBP, including a signal peptide of 19 residues, a pro-peptide of 7 amino acids, a mature portion of 222 amino acids and a C-terminal extension of 3 amino acids. This plasmid formed was named pSX556.

In order to prepare recombinant baculovirus expressing HBP, the MAXBAC kit from Invitrogen Corp. (San Diego, Calif.) Was used and all manipulations were performed according to the Baculovirus Expression System Manual (Version 1.5.5). Was performed. Briefly, 1 μg linear AcMNPV DNA and 3 μg pSX556 were cotransfected with Spodoptera frugiperda (SF9) insect cells (2 × 10 ⁶ cells in 60 mm dish). The obtained culture supernatant was recovered after 7 days. Monolayers of fresh SF9 cells in 100 mm plates were infected with various diluted virus supernatants and then 1.5% agarose was applied on complete TNM-FH medium containing 150 μg / ml of X-gal. After 8 days, six putative recombinant plaques were identified in blue color and used to infect 6 well plates containing SF9 cells. After 5 days, the corresponding viral DNA was purified and PCR reactions were performed using forward and reverse primers next to the recombination site in the viral DNA. After identifying the PCR product on the agarose gel, repeated experiments of plaque purification were performed once more to confirm that the corresponding purest virus was free of wild type virus in the final recombinant virus stock. Production of recombinant HBP was carried out using insect cells (SF9 and SF21) adapted for growth in SF900-II medium (Gibco BRL / Life-Technologies) containing no serum. Generally, 5 L spinner incubator or 10 L fermenter with cell density of 1 × 10 ⁶ / mL was infected with 1 MOI and the medium was recovered 3 days after infection. Purification of HBP was performed as described in US Pat. No. 5,814,602.

HBPs from insect cells infected with recombinant baculovirus were examined by SDS-PAGE. This HBP has a slightly higher molecular weight than the original HBP purified from human blood. When the N-terminal sequence was determined, it was confirmed that almost 100% of the material produced contained pro-peptides consisting of seven amino acids in front of the mature part, in the same way as human myeloid neutrophil precursor cells in the bone marrow. It indicates that it is not possible to produce pro-HBP.

Example 2: Expression of Δpro-HBP in Insect Cells

The oligonucleotide linker (shown below) contains the first 4 amino acids of the signal peptide and the mature HBP, but includes the first 99 bp of the HBP sequence (BamH to EagI) excluding the portion corresponding to the proregion (73-87). PSX559 was prepared by substituting the original Bam HI- Eag I in pSX556.

The linker consists of four oligonucleotides annealed in pairs to provide the following two duplexes.

MHJ2568 / LWN5746:

5'-GATCCACCATGACCCGGCTGACAGTCCTGGCCC-3 '(SEQ ID NO .: 17)

3'-GTGGTACTGGGCCGACTGTCAGGACCGGGACGACC-5 '(SEQ ID NO .: 18)

LWN5745 / MHJ2566:

5'-P-TGCTGGCTGGTCTGCTGGCGTCCTCGAGGGCCATCGTTGGC-3 '(SEQ ID NO .: 19)

3'-GACCAGACGACCGCAGGAGCTCCCGGTAGCAACCGCCGG-5 '(SEQ ID NO .: 20)

SF9 cells were transfected with recombinant baculovirus and the expressed HBP was purified as described in Example 1 above. After determining the N-terminal sequence, (sequencing) now proceeds 90% starting with Ile ¹ , with the remaining 10% going further downstream starting with Arg ⁵ .

To compare the expression of pro-HBP and HBP, time course experiments were performed. After infection, culture medium aliquots were taken daily for the first 6 days (after 5 days) and tested by HBP-specific ELISA. The mean value (n = 3) of the maximum pro-HBP yield (day 4) is 100%. It was found that HBP is expressed 2-3 times less than the pro-HBP type. Optimal yield of pro-HBP was obtained 4 days after infection, but the remaining yield of HBP remained almost unchanged for 3-4 days.

Electrospray mass spectrum (ESMS) analysis of recombinant HBP shows that the molecular weight is 27,237 + 3. The calculated value for the 225 amino acid HBP type (plus the three amino acid C-terminal extension to the mature portion) is 24,268.6. This is because HBP contains three potential glycosylation sites (Asn ¹⁰⁰ , Asn ¹¹⁴ and Asn ¹⁴⁵ ). This corresponds to 2,968, the mass of the glycan moiety (s). This in turn corresponds to the theoretical values of two Man3 [Fuc] GIcNAc2 units and one Man3GIcNAc2 unit.

Example 3: Expression of Pro-HBP in HEK 293 Cells

The following method was used to prepare an expression vector transfected into HEK 293 cells. First, plasmid pSX556 was prepared according to the method described in Example 1.

pSX556 was prepared using primers PBRa 246 5'-CCGGGGATCCAACTAGGCTGGCCCCGGTCCCGG-3 '(SEQ ID NO: 21) with Pfu polymerase according to the manufacturer's (Stratagene) instructions.

PBRa 247 5'-CCGGGGATCCGATGACCCGGCTGACAGTCCTGG-3 '(SEQ ID NO: 22) is used as a template in a PCR reaction. After cutting the PCR product with Bam HI, the linearized fragment was mammal with Bam HI by ligation in the correct orientation in the animal expression vector pcDNA3 (Invitrogen) was prepared pcDNA3-HBP.

The following method was used to transfect HEK cells. The day before infection, ⁵ × 10 ⁵ HEK 293 cells were seeded in 10 cm dishes containing 10 ml of DMEM, 10% FCS and penicillin / streptomycin. 20 g of pcDNA3-HBP were transfected HEK 293 cells by modified calcium phosphate method (Chen and Okayama, 1987, Molecular and Cellular Biology 7: 2745 2752). Transfectants were selected with 600 g / ml Geneticin (Life Technologies). The expression of HBP was tested using the HBP specific sandwich-ELISA method for the primary transfection group mixed into one. The shape of the transfected cells is similar to HEK 293 cells that are not transfected. Transfection-populations are subcloned by defined dilution methods and the best clones (1 / E-11) can produce 11.5 g HBP / ml / day on a T-25 flask scale. In particular, 300 cells were seeded in 5 × 96 well plates and expression assays were performed on cell clones (subclones) derived from one cell.

To characterize the produced HBP material, clone 1 / E-11 was grown by monolayer culture in serum-free medium for 6 days with fresh medium changes every day. The HBP supernatant concentration is 8 mg / l as determined by integrating the area from HPLC (using baculovirus HBP as a standard). Agarose linked aprotinin was used for purification and eluted with 1M NaCl. This single step purification gives 99% pure sample as determined by HPLC analysis. When the N-terminal sequence was determined, it was confirmed that almost 100% of the material produced contained pro-peptides consisting of seven amino acids in front of the mature part, in the same way as human myeloid neutrophil precursor cells in the bone marrow. It indicates that it is not possible to produce pro-HBP. As described in Example 1, the same result was obtained when HBP was expressed in a baculovirus / insect cell system. The molecular weight was determined to be 31,693 by MALDI, so the glycan moiety has a mass of 6,755. Many other possibilities are essential, assuming that all three N-linked glycosylation sites in this mass correspond to three di-sialyated, galactosylated biantennary structures Although possible, in all HEK 293 cells, HBP appears to contain complex natural N-linked oligosaccharides.

Example 4: Expression of Mature HBP in HEK 293 Cells

The following method was used to prepare expression vectors for transfecting HEK 293 cells. First, plasmid pSX559 was prepared according to the method described in Example 2.

pSX559, with Pfu polymerase according to the manufacturer's (Stratagene) instructions, includes the following primers; PBRa 246 5'-CCGGGGATCCAACTAGGCTGGCCCCGGTCCCGG-3 '(SEQ ID NO: 21) and PBRa 247 5'-CCGGGGATCCGATGACCCGGCTGACAGTCCTGG-3' (SEQ ID NO: 22) are used as templates. The PCR reaction product was digested with Bam HI, and the fragments were ligated in the correct orientation to the mammalian expression vector pcDNA3 (Invitrogen) linearized with BamHI to prepare pcDNA3-HBPΔPro.

pcDNA3-HBPΔpro was transfected into HEK 293 cells, and the expression-positive expression of HBP was tested by the HBP-ELISA method for the transfection-population mixed into one. HEK 293 cells are transfected using the following method. The day before infection, ⁵ × 10 ⁵ HEK 293 cells were seeded in 10 cm dishes containing 10 ml of DMEM, 10% FCS and penicillin / streptomycin. 20 g of pcDNA3-HBPΔpro were transfected into HEK 293 cells by modified calcium phosphate method (Chen and Okayama, 1987, Molecular and Cellular Biology 7: 2745 2752). The transfectants were selected with 600 g / ml Genithinin (Life Technologies). Positive expression of HBP was tested using the HBP specific sandwich-ELISA method for the initial transfection-population mixed into one. The shape of the transfected cells is similar to HEK 293 cells that are not transfected. Transfection-populations are subcloned by defined dilution methods. In particular, 300 cells were inoculated into five in 96 well plates, and cell clones (subclones) derived from one cell were tested for expression. The best clone 3 / E-4 (3.25 g HBP / ml / day) selected by defined dilution method was isolated.

To characterize the produced HBP, clone 3 / E-4 was grown by monolayer culture in serum-free medium, with daily change of medium. The medium was filtered using Sartorius filters and purified on a CM-Sepharose fast flow column by the method described above. 0.2 mg of pure HBP was isolated. 10 μg was further purified by small scale preparative HPLC for N-terminal sequencing and mass identification. 95% of purified HBP contains the right N-terminal sequence (IVGGRKARPRQFPFL, SEQ ID NO: 23). However, the remaining 5% showed truncation starting at amino acid number 5 (RKARPRQFPFLASIQN, SEQ ID NO: 24). This N-terminal truncation form is consistent with the baculovirus / insect cell HBP results described in Example 1. Matrix assisted laser desorption ionization (MALDI) and electrospray mass spectroscopy (ESMS) mass detection showed two broad peaks with an average mass of 30,550. This indicates that the mass of the glycan moiety is approximately 6,300.

Example 5: Biological Activity of Component HBP Produced by HEK 293

In order to compare the biological activity of mature HBP produced by HEK 293 cells with insect cell-derived mature HBP, both recombinants were examined by monocyte / IL6 assay: human mononuclear cells were buffy-normal blood. coats). In 24-well plates, 2 × 10 ⁵ cells per well were seeded in 1 ml of DMEM medium containing 2 mM Glutamax, 1% non-essential amino acid and 1 mg / ml BSA. LPS (E. coli derived, Sigma) and recombinant HBP were treated as described in Table 1. After incubation for 24 hours at 37 ° C. (5% CO ₂ ), the cell supernatants were recovered by centrifugation and clarified and then stored at −20 ° C. until used for analysis of their IL-6 content. IL-6 determinations were performed using the Biotrek ELISA system (Amersham Pharmacia Biotech).

As can be seen in Table 1, LPS that induces IL-6 release increases with increasing amount of insect-HBP, however, if only 2 g of HEK 293-HBP is present, it induces IL-6 release. LPS was nearly identical to that of 10 g of insect-HBP. This indicates that the specific activity of HEK 293 mature HBP is about 5 times higher than the specific activity of insect derived mature HBP. This increase in specific activity is most likely due to the many complex N-linked glycosylation patterns performed by mammalian HEK 293 cells.

+/- 10ng LPS IL-6 (pg / ml) Monocytes alone - 920 + 3575 + 2g Insect-HBP - 890 + 4200 +5 g insect-HBP - 1120 + 8050 +10 g insect-HBP - 1575 + 11800 +2 g HEK 293-HBP - 1000 + 12550

Example 6: Glucose Consumption and Lactate Production in HEK Cell Lines Producing HBP

To determine how metabolism of stable HEK 293 transformants producing HBP is affected by the HBP product itself, three different HEK 293 cell lines [1] HEK 293 cell line (control); 2) 1 / C-6 (clone produced in exactly the same manner as 1 / E-11 in Example 3 producing pro-HBK); 3) The consumption of glucose and the production of lactate were measured using the KODAK EKTACHEM DT 60II instrument at 3 / E-4 (clone described in Example 4 producing mature HBP). The following two media were used: DMEM (Dulbecco's Modified Eagle Medium) containing 4500 mg / L glucose (Life Technologies) and 10% FCS and no synthetic medium.

The results are shown in Table 2.

Glucose (mg / L) Lactate (mg / ℓ) Fresh DMEM Badge 3690 153 After 20 hours incubation HEK 293 2358 1215 1 / C-6 1836 1620 3 / E-4 1296 1935 Medium without fresh protein 4158 63 After 4 hours incubation HEK 293 3888 450 1 / C-6 3654 540 3 / E-4 3258 855 After 18 hours of incubation HEK 293 2448 1440 1 / C-6 3 / E-4 1926 1305

As shown in Table 2, glucose consumption and lactate production in both media are increased in HEK 293 cell lines producing HBP. In addition, 3 / E-4 cell lines producing mature HBP had the highest glucose consumption and lactate production in both DMEM medium and protein-free medium after incubation for 4 hours. After 18 hours, the lactate concentration in the medium containing no protein of 3 / E-4 cells is the smallest due to the breakdown of lactate.

The results shown in Table 2 indicate that in some cases when HBP is present around the mitochondria in HEK 293 cells producing HBP, HBP prevents ATP production of oxygen dependent mitochondria. As a result, due to their embryonic origin, ATP production occurs only by anaerobic glycolysis in HEK 293 cells.

Example 7: Construction of pDC312-HBPΔpro

The following method was used to construct pDC312-HBPΔpro. First, plasmid pSX559 was produced by the method described in Example 2. pSX559 was prepared with the following primers with PfX polymerase according to the manufacturer's instructions; Used as template in PCR reactions with ViLi118 5'-CCGGGATCCTAGTCCCACCATGACCCGGCTGACA-3 '(SEQ ID NO: 25) and ViLi119 5'-GCGCGCGGCCGCCTAGGCTGGCCCCGG-3' (SEQ ID NO: 26). The prepared PCR fragment was digested with restriction enzymes Bam HI and Not I. The cleaved fragment was gel-purified and ligated in the correct orientation to the mammalian expression vector pDC312 (Immunex) digested with Bam HI and Not I, resulting in pDC312-HBPΔPro.

pDC312-HBPΔpro was transfected into HEK 293 cells, and the transfection-population fused to one was tested for expression of HBP by the use of HBP-ELISA. The following method was used to transfect HEK 293 cells. The day before infection, ⁵ × 10 ⁵ HEK 293 cells were seeded in 10 cm dishes containing 10 ml of DMEM, 10% FCS and penicillin / streptomycin. 20 g of pDC312-HBPΔpro were transfected HEK 293 cells by modified calcium phosphate method (Chen and Okayama, 1987, Molecular and Cellular Biology 7: 2745 2752). Transfectants were selected with 0.25M methotrexate (MTX) (LifeTechnologies). The expression of HBP was examined using HBP specific sandwich-ELISA for the initial transfection-group mixed into one. The shape of the transfected cells is similar to HEK 293 cells that are not transfected. Transfection-populations were subcloned by defined dilution methods. In particular, 300 cells were inoculated in five in 96 well plates, and expression was tested for cell clones (subclones) derived from one cell. The best clone 3 / E-4 (9.4 g HBP / ml / day) selected by defined dilution method was isolated.

To characterize the produced HBP, clone 3 / E-4 was grown monolayered in serum-free medium with fresh medium changes every day. The medium was filtered using a Sartorius filter and purified on a CM-Sepharose fast flow column, according to the method described above. 0.2 mg of pure HBP was isolated. Further 10 μg purification was performed by small scale preparative HPLC for N-terminal sequencing and mass identification. 95% of purified HBP contains the right N-terminal sequence (IVGGRKARPRQFPFL, SEQ ID NO: 27). However, the remaining 5% appeared truncated starting at amino acid number 5 (RKARPRQFPFLASIQN, SEQ ID NO: 28). This N-terminal truncation form is consistent with the baculovirus / insect cell HBP results described in Example 1. MALDI and ESMS mass detection showed two broad peaks with an average mass of 30,550. This indicates that the mass of the glycan moiety is approximately 6,300.

Example 8: Hollow-Fiber Cell Culture System

293 cells (4.037 x 10 ² ), i.e., HBP 293 3 / B-5 # / 0, are hollow fiber cartridges (two bioreactors) of an automated cell culture system (AcuSyst Maximazer, CELLEX Biosciences, INc., Minneapolis, USA) , Incubated in Spinner bottles (Techne, Cambridge, UK) before inoculation into extra capillary (EC) space of 10 Kd mol wt cleavage, 1,5 m 2 (SP 16/10) . Examination of the cell line confirmed the absence of mycoplasma (GEN-PROBE, GEN-Probe incorporated, San die, USA). Start control parameters were as follows: temperature = 36.5 ° C .; pH = 7.10; Oxygen pressure = 120 Hg / mm. 293 SFM III medium (Life Technologies, Paisiey, UK) with 3.5 mM glutamine (Life Technologies, Paisiey, UK) containing dissolved gas was pumped at 250 ml / min through the interior of the fiber (circulating pump) And flowed across the membrane into the EC space. The medium flow rate was 50 ml / hr, and 100 ml / hr after one day of culture; 200 ml / hr after 2-20: 08 days (2 days 20 hours 8 minutes); 250 mL / hr after 3-19: 40 days (3 days 19 hours 40 minutes); At 300 ml / hr after 4-20: 39 days (4 days 20 hours 39 minutes); 400 ml / hr after 8-23: 15 days (8 days 23 hours 15 minutes); 450 mL / hr after 19-18: 41 days (19 days 19 hours 41 minutes), 500 mL / hr after 22-01: 16 days (22 days 1 hour 16 minutes), 24-18: 46 days (24 550 ml / hr after 18 hours and 46 minutes), and 600 ml / hr after 24-19: 06 days (24 hours and 19 hours and 6 minutes); It increased to 650 ml / hr after 30-20: 43 days (20 hours 43 minutes on 30 days) and 700 ml / hr after 33-01: 03 (1 hour 3 minutes on 33 days). Continuous recovery and feed of the EC chamber started at 2 ml / hr after 8-22: 59 days. The operation was terminated after 34-23: 01 days and a total of 1,529 mL of recovery was collected.

The following method was used to purify HBP from HEK293 suspension. The pH of the suspension was adjusted to 8.5 with 1N sodium hydroxide and then filtered through a GF / A filter. The column (SP 16/10) is equilibrated with 30 mM Tris (pH 8.5) and the flow rate is 4 ml / min. Each condition was applied and washed with equilibration buffer. HBP was eluted with a NaCl concentration gradient. HBP eluted at about 0.9 M NaCl. Each population fraction was collected and quantified on a C4 assay column.

The results are shown in Table 3.

Batch number Suspension volume (ml) Suspension Concentration (g / l) Total amount (mg) in suspension Total amount in the mass fraction (mg) yield(%) 000301 260 0.3 78 65 83 8 / 3-2000 155 0.4 62 50 81 8 / 3-2000 200 0.4 80 60 75 000314 150 0.3 45 80 177 000314 130 0.3 39 55 141 000315 34 - - About 5 - 000317 150 0.3 45 50 111 000320 165 0.3 50 50 100

About 400 mg was collected as a whole. The concentration of HBP was 0.3-0.4 g / l.

All batches showed the same results in preliminary purification and HPLC analysis. The first batch was further analyzed for molecular weight and sequence. The molecular weight is 30,5 kD and some dimers have also been found. The sequence is related to the expected HBP (15 acts).

SDS analysis showed strong bands at 36 kD and two weak bands at 10-20 kD.

The invention described and claimed herein is not to be limited in scope by the specific embodiments described herein. These examples are intended to illustrate some aspects of the invention by way of example. Any equivalent embodiments may be presented without departing from the scope of the present invention. Indeed, various modifications of the invention, as well as those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications should also be presented within the scope of the present invention.

Various documents are cited here, the disclosure of which is incorporated into the document as a whole.

According to this method, mature heparin-binding proteins can be obtained in high yield by a simple but efficient method.

Claims (22)

(a) introducing a nucleic acid encoding a mammalian heparin-binding protein (HBP) into a mammalian cell; (b) culturing the cells of step (a) under conditions inducing expression of HBP; And (c) recovering the HBP from the culture medium A method for producing a heparin-binding protein in a mammalian cell that can be cultured under anaerobic conditions after introducing a nucleic acid encoding a heparin-binding protein into the cell. The method of claim 1, wherein the mammalian heparin-binding protein is human or swine HBP. The method of claim 1, wherein the HBP recovered from the culture medium has an amino acid sequence having at least about 80% homology with the amino acid sequence set forth in SEQ ID NO: 1 or 2 or alleles or natural variants thereof. The compound of claim 1, comprising: (i) the sequence set forth in SEQ ID NO: 3, 4, 5, 7, 9 or 11; (Ii) its complementary strands; Or (iii) introducing into said mammalian cell a nucleic acid sequence which forms a hybrid with the subsequence of (a) or (b). The method of claim 1, wherein the HBP recovered from the culture medium has the amino acid sequence of SEQ ID NO: 1 or 2. The method according to claim 1, wherein the nucleic acid sequence set forth in SEQ ID NO: 3, 4, 5, 7, 9 or 11 is introduced into the mammalian cell. The method of claim 1, wherein said nucleic acid sequence encodes a mature HBP present upstream of a signal sequence. The nucleic acid sequence of claim 1, wherein the cell of step (a) comprises a nucleic acid sequence encoding a heparin-binding protein pro-sequence and a mature heparin-binding protein and encodes an N-terminally expanded heparin-binding protein. And the nucleic acid sequence encoding the protease cleavage site to be located between the nucleic acid sequence encoding the N-terminal extension and the nucleic acid sequence encoding the mature heparin-binding protein. 8. The method of claim 7, further comprising cleaving the N-terminally extended HBP to obtain mature HBP. The method of claim 1, wherein the cell of step (a) comprises a nucleic acid sequence encoding a heparin-binding protein signal sequence, heparin binding pro-sequence, mature heparin binding protein and heparin-binding protein pro-sequence. And a nucleic acid sequence encoding a protease cleavage site to be located between the nucleic acid sequence encoding and the nucleic acid sequence encoding the mature heparin-binding protein. 10. The method of claim 9, further comprising cleaving the N-terminally expanded HBP to obtain mature HBP. The method of claim 1, wherein the host cell is an adenovirus transformed cell. The method of claim 1, wherein the host cell is a cell derived from an embryo. The method of claim 1, wherein the host cell is a human embryonic kidney cell. The method of claim 1, wherein the host cell is a human embryonic kidney 293 cell. The method of claim 1, further comprising purifying the heparin-binding protein. (a) introducing a nucleic acid encoding a heparin-binding protein into mammalian host cell (s) that can be cultured under anaerobic conditions after introduction of the nucleic acid; (b) selecting a mammalian host cell of step (a) comprising a nucleic acid encoding a heparin-binding protein; (c) culturing the host cell in a serum-free medium and optionally a medium containing no protein; And (d) isolating the heparin-binding protein. The method of claim 16, further comprising purifying the heparin binding protein. A recombinant mammalian host cell comprising a nucleic acid sequence encoding the heparin binding protein, which may be cultured under anaerobic conditions after introduction of a nucleic acid encoding a heparin-binding protein. 19. The recombinant mammalian host cell of claim 18, wherein said nucleic acid sequence encodes a human heparin-binding protein. 19. The recombinant mammalian host cell of claim 18, wherein the nucleic acid sequence encodes a mature HBP present upstream of the signal sequence. 19. The recombinant mammalian host cell of claim 18, wherein the recombinant host cell is a human embryonic kidney 293 cell.