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WO2003080660A2 - Procede de preparation d'une proteine de liaison a l'heparine (hbp) mammifere recombinee - Google Patents

Procede de preparation d'une proteine de liaison a l'heparine (hbp) mammifere recombinee Download PDF

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Publication number
WO2003080660A2
WO2003080660A2 PCT/DK2003/000207 DK0300207W WO03080660A2 WO 2003080660 A2 WO2003080660 A2 WO 2003080660A2 DK 0300207 W DK0300207 W DK 0300207W WO 03080660 A2 WO03080660 A2 WO 03080660A2
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hbp
fusion protein
polypeptide
protein
cleavage site
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PCT/DK2003/000207
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WO2003080660A3 (fr
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Helle Fabricius WÖLDIKE
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Leukotech A/S
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Priority to AU2003226907A priority Critical patent/AU2003226907A1/en
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Publication of WO2003080660A3 publication Critical patent/WO2003080660A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • HBP heparin-binding protein
  • the present invention relates to methods of making heparin-binding protein (HBP) in a recombinant bacterial expression system.
  • this invention relates to the preparation of HBP from an insoluble fusion protein expressed in bacterial cells and accumulated in inclusion bodies in the cytoplasm of bacterium.
  • the invention further relates to methods of separation of HBP from a fusion partner polypeptide, purification of HBP from said polypeptide, and refolding of HBP.
  • a local infection or injury in any tissue rapidly attracts white blood cells into the affected region as part of the inflammatory response, which helps fight the infection or heal the wound.
  • an initial wave of inflammatory cells comprised primarily of polymorphonuclear leukocytes (PMNs) is soon followed by a second wave of cells, which are predominantly monocytes.
  • PMNs polymorphonuclear leukocytes
  • the preferential migration of monocytes during the latter phase of inflammation indicates the requirement for highly cell-specific chemoattractants, which have little or no effect on the migration of PMNs.
  • Accumulating evidence indicates that a protein isolated from human PMNs can be a candidate for the role of a monocyte-specific chemoattractant.
  • HBP human heparin-binding protein
  • pHBP porcine heparin-binding protein
  • HBP HBP has otherwise been termed CAP37 (WO 91/00907, US 5,458,874 and 5,484,885) and azurocidin (CG. Wilde et al. 1990, J. Biol. Chem. 265:2038-41 ).
  • HBP has far reaching and important functions involving the cellular progression, antimicrobal and antineoplastic defences of the host, however, the usage of purified HBP as isolated from PNMs may be limited because of (1) the very small quantities that can be purified and (2) the potential hazards of using blood products, especially of human origin. Use of recombinant HBP may overcome these problems.
  • HBP has been produced via recombinant DNA methods in insect cells. (Rasmussen et al., FEBS Lett 1996, 390:109-112). The protein has also been produced in in hu- man kidney 293 cells (Alberdi et al. 1997, FASEB J 11 :1915 and in RBL (rat baso- philic leukaemia) cells (disclosed in PCT /VO00/66627).
  • the patent describes an example of production of a HBP using a gluta- thione S-transferase (GST) gene fusion expression system that was developed by Smith and Johnson (Gene 1988, 67:31-40) to direct the synthesis of foreign poly- peptides in Escherichia coli (E. coli).
  • GST gluta- thione S-transferase
  • E. coli Escherichia coli
  • fusion polypep- tides retain, in the majority of cases, soluble in the cytoplasm of bacteria, and can be purified from crude bacterial lysates by affinity chromatography on immobilised glu- tathione.
  • the patent does not disclose neither the appearance of the fusion HBP-
  • HBP heparin-binding protein
  • the present invention provides a method for the preparation of an insoluble fusion protein comprising a heparin-binding protein (HBP), a cleavage site, and a second polypeptide in recombinant bacterial cells comprising a) providing a recombinant expression vector including a DNA construct encoding a fusion protein, wherein said fusion protein comprises HBP, a second polypeptide and a protease cleavage site;
  • HBP heparin-binding protein
  • step (a) transforming host cells of bacterium with the recombinant vector of step (a);
  • step (b) culturing the transformed host cells of step (b) which express and accumulate the fusion protein of step (a) in insoluble form in the cytoplasm;
  • step (c) lysing the cells of step (c);
  • step (d) obtaining a precipitate comprising the fusion protein in the insoluble fraction of the host cell lysate of step (d).
  • the invention provides a method for producing of recombinant heparin-binding protein (HBP) in bacterial cells comprising
  • step (a) transforming host cells of bacterium with the recombinant vector of step (a);
  • step (b) culturing the transformed host cells of step (b) which express and accumulate the fusion protein of step (a) in insoluble form in the cytoplasm;
  • step (c) lysing the cells of step (c);
  • step (d) obtaining the expressed fusion protein in the insoluble fraction of the host cell lysate of step (d);
  • step (e) dissolving the obtained fusion protein of step (e) in aqueous solution
  • the invention provides a method for producing recombinant heparin-binding protein (HBP) in bacterial cells comprising,
  • step (b) cleaving the solved fusion protein of step (b);
  • step (c) purifying HBP after the cleavage of step (c);
  • step (d) optionally refolding HBP obtained after purification of step (d).
  • the invention provides a method for producing in bacterial cells a mammalian heparin-binding protein (HBP) comprising,
  • An additional aspect of the present invention resides in providing a recombinant expression vector including a DNA construct comprising a DNA sequence encoding the gene of a mammalian HBP, allelic or natural variants thereof, which is fused in frame to a DNA sequence encoding a protease cleavage site, which in turn is fused in frame to a DNA sequence encoding a second polypeptide.
  • Another additional aspect of the present invention resides in providing a DNA construct comprising a DNA sequence encoding the gene of a mammalian HBP, or allelic or natural variants thereof, which is fused in frame to a DNA sequence encoding a protease cleavage site, which in turn is fused in frame to a DNA sequence encoding a second polypeptide.
  • the invention is further directed to the construction of a fusion protein comprising an amino acid sequence of HBP, an amino acid sequence of a second polypeptide and an amino acid sequence of a protease cleavage site, said amino acid sequence of the protease cleavage site being positioned between the amino acid sequence of HBP and the amino acid sequence of the second polypeptide, wherein the second polypeptide provides the fusion protein with capabilities of forming insoluble aggregates in cytoplasm of bacteria after being expressed in said bacteria.
  • pHW1295 encodes the mature human HBP fused to glutathione-S-reductase (GST) via the factor Xa protease cleavage site
  • pHW1296 encodes the precursor human HBP fused to GST via the thrombin cleavage site (Example 3).
  • Fig. 4 Outline of construction of the expression plasmids for the HBP-thioredoxin fusion proteins with the enterokinase cleavage site, pHW1288 and pHW1289, encoding the mature human HBP with and without N-terminal methionine respectively, and pHW1290 encoding the precursor human HBP (Example 4).
  • Fig. 5 Outline of construction of the expression plasmid pHW1306 encoding the mature human HBP fused to thioredoxin via the Achromobacter lyticus protease cleavage site.
  • the sequence of HBP carries a point mutation K6R (Example 5).
  • Fig. 6 Outline of construction of the expression plasmid pHW1383 encoding the mature human HBP fused to ubiquitin via the Achromobacter lyticus protease cleavage site.
  • the sequence of HBP carries a point mutation K6R (Example 6).
  • Fig. 7 Proteins expressed in recombinant E. coli transformed with the expression plasmids a- pHW1280 (lanes 1, 2, 3), pHW1283 (lanes 4, 5, 6), pHW1288 (lanes 7, 8, 9), pHW1289 (lanes 10, 11, 12), pHW1290 (lanes 13, 14, 15), pHW1306 (16, 17,
  • the transformed cells were grown without induction of the protein expression ( ⁇ lanes, whole cell lysates), or were induced for 3h before lysis.
  • the lysates of induced cells were centrifuged at 10, 000 g for 10 min (P- pellets, and S- supernatants ).
  • HBP HEPARIN-BINDING PROTEIN
  • HBP HBP a protein i) showing at least one of the following biological activities: (1) chemotactic activity for monocytes; (2) bacterial lipopolysaccharide-binding (LPS) activity; (3) antibiotic activity; (4) antiapoptotic activity,
  • HBP is produced in the azurophil granules of polymorphonuclear leucocytes.
  • Full length HBP has in glycosylated form a molecular weight of about 32 kD as determined by SDS-PAGE under reduced conditions.
  • HBP is a protein having preferably at least two of the above activities, such as in a more preferred embodiment at least tree of the above activities, such as in the most preferred embodiment at least four of the above activities.
  • the present invention is directed to a method for preparation of recombinant HBP.
  • the amino acid sequence of recombinant HBP may suitably be of mammalian, in particular human or porcine HBP.
  • HBP is mature human HBP which has at least about an 80% identity with the amino acid sequence set forth in SEQ ID NO: 1 , more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97% (hereinafter "homologous polypep- tides").
  • HBP is a mature porcine HBP which has at least about an 80% identity with the amino acid sequence set forth in SEQ ID NO: 11 , more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%.
  • HBP may also have at least 80% identity with the amino acid se- quence set forth in SEQ ID NO: 3 (a human HBP including the sequence of mature protein and the pro sequence), SEQ ID NO:5 (a human HBP including the sequence of mature protein, and pro and signal sequences), SEQ ID NO:7 (a human HBP including the sequence of mature protein and the signal sequence and), SEQ ID NO:9 (a mature human HBP wherein the lysine at position 6 is replaced with argin- ine), SEQ ID NO:13 (a porcine HBP including the sequence of mature protein and pro sequence), SEQ ID NO: 15 (a porcine HBP including the sequence of mature protein, and pro and signal sequences), and SEQ ID NO: 17 (a porcine HBP including the sequence of mature protein and the signal sequence)
  • SEQ ID NO: 3 a human HBP including the sequence of mature protein and the pro sequence
  • SEQ ID NO:5 a human HBP including the sequence of mature protein, and pro and signal sequences
  • SEQ ID NO:7 a
  • a “homologous polypeptide” is defined as a polypeptide comprising one of the above amino acid sequences and showing at least one of the following biological activities: (1) chemotactic activity for monocytes; (2) bacterial lipopolysaccharide- binding activity; (3) antibacterial activity; (4) antiapoptotic activity, (5) capability of binding an antibody, said antibody being raised against full length HBP, more pref- erably at least two of above activities, even more preferably at least three of above activities, and the most preferably at least four of above activities.
  • amino acid sequences of the homologous polypeptides differ from the amino acid sequence set forth in SEQ ID NOS: 1 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 mi- nor 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 an- other function, such as a polyhistidine 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), hydro- phobic 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. Hill, 1979, in, The Proteins, Academic Press, New
  • the recombinant heparin binding protein may be encoded by a nucleic acid sequence having at least about 60% identity with the nucleic acid sequence set forth in SEQ ID NO: 2 (which encodes a mature human HBP depicted in SEQ ID NO: 1), SEQ ID NO: 4 (which encodes a human HBP which includes the pro sequence and the sequence of mature protein, depicted in SEQ ID NO: 3), SEQ ID NO: 6 (which encodes a human HBP which includes the signal sequence, pro sequence and sequence of the mature protein, depicted in SEQ ID NO: 5), SEQ ID NO:8 (which encodes a human HBP which includes the signal sequence and sequence of the mature protein, depicted in SEQ ID NO:7), SEQ ID NO: 10 (which encodes the mature human HBP having the lysine residue at position 6 replaced for arginine, depicted in SEQ ID NO:9), or SEQ ID NO: 12 (which encodes the mature porcine HBP, depicted in SEQ ID NO:
  • 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, J. Mol. Biol. 48:443-453). For purposes of determining the degree of identity between two nucleic acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.
  • the analogous sequence may be constructed on the basis of the nucleic acid sequence presented as the HBP encoding part of SEQ ID NOS:1 , 3, 5, 7, 9, 11 , 13, 15 or 17, e.g., a sub-sequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of 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 mutagene- sis (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.
  • the carrier material is washed three times each for 30 minutes using 2X SSC, 0.2% SDS preferably at least at 50°C (very low stringency), more preferably at least at 55°C (low stringency), more preferably at least at 60°C (medium stringency), more preferably at least at 65°C (medium-high stringency), even more preferably at least at 70°C (high stringency) and most preferably at least at 75°C (very high stringency).
  • the fusion protein DNA construct and recombinant expression vector
  • a method for preparation of recombinant HBP comprises providing a recombinant expression vector including a DNA construct encoding a fusion protein, wherein said fusion protein comprises HBP, a second polypeptide and a protease cleavage site.
  • the nucleic acid sequence encoding the fusion protein may be prepared synthetically by established standard methods, e. g. the phosphoamidine method (Beau- cage and Caruthers, 1981 , Tetrahedron Lett. 22:1859-1869), or the method de- scribed by Matthes et al. (EMBO J., 1984, 3: 2021-2028).
  • the techniques used to isolate or clone a nucleic acid sequence encoding HBP and the other proteins used in the method of the present invention are well-known in the art and include isolation of genomic DNA and preparation of cDNA, or a combination thereof. Cloning the nucleic acid sequences of the present invention from such genomic DNA can be effected, e. g. by using the polymerase chain reaction (PCR) or antibody screening of the expression libraries to detect the cloned DNA fragments with sheared structural features (Innis et al. A Guide to Methods and Applications. Academic Press, NY 1990.). Other nucleic acid amplification procedures such as the ligase chain reaction (LCR), ligation activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used as well.
  • LCR ligase chain reaction
  • LAT ligation activated transcription
  • NASBA nucleic acid sequence-based amplification
  • a nucleic acid sequence of the invention comprises a DNA sequence encoding HBP, wherein said sequence is a nucleic acid sequence which hybridises to a sequence capable of hybridising to the nucleic sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18, or allelic or natural variants thereof, a DNA sequence encoding a protease cleavage site, and a DNA sequence encoding a second polypeptide.
  • the DNA sequence encoding a second polypeptide is a sequence which hybridises to the DNA sequence of calf chymosin, or allelic or natural variants of thereof.
  • the DNA sequence encoding a second polypeptide is a sequence which hybridises to the DNA sequence of bacterial thioredoxin, or allelic or natural variants of thereof.
  • the DNA sequence encoding a second polypeptide is a sequence, which hybridises to the DNA sequence of ubiquitin.
  • the DNA sequence encoding a protease cleavage site may be represented by a DNA sequence encoding the polypeptide with an amino acid sequence of the either thrombin, or enterokinase, or Factor Xa, or Achromobacter lyticus protease cleavage site.
  • the DNA sequences encoding partner polypeptides of the fusion protein are fused in frame giving a DNA construct encoding the fusion protein.
  • the nucleic sequences of said DNA construct are fused in frame in the following order: a second polypeptide DNA sequence precedes the sequence of a protease cleavage site, which, in turn, precedes the DNA sequence encoding HBP.
  • nucleic acid sequence encoding the fusion protein of the invention is then in- serted into a recombinant expression vector.
  • the recombinant expression vector may be any vector that may conveniently be subjected to recombinant DNA procedures.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i. e. a vector which exists as an extra chromosomal entity, 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.
  • nucleic acid sequence encoding the fusion protein of the invention should be linked to a suitable promoter sequence.
  • the promoter may be any nucleic acid sequence, which shows transcriptional activ- ity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the nucleic acid sequence encoding the HBP-fusion protein, especially in a bacterial host cell are the promoters obtained from the E.
  • nucleic acid sequence encoding the fusion protein of the invention may be op- erably connected to a suitable terminator of transcription.
  • a nucleic acid sequence encoding the fusion protein comprising HBP, a second polypeptide and a protease cleavage site is inserted into the vector between the promoter and terminator regions in the following order: promoter-second polypeptide-cleavage site-HBP-terminator.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • sequences enabling the vector to replicate are various replication origins sequences.
  • the vector may also comprise a selectable marker, e. g. a gene, the product of which confers resistance to a drug, e. g. ampicillin, kanamycin, tetracycline, chlor- amphenicol.
  • a selectable marker e. g. a gene, the product of which confers resistance to a drug, e. g. ampicillin, kanamycin, tetracycline, chlor- amphenicol.
  • the fusion protein a fusion partner polypeptide and cleavage site
  • a fusion protein comprises an amino acid sequence of HBP, an amino acid sequence of a second polypeptide and an amino acid sequence of the protease cleavage site, said amino acid sequence of the protease cleavage site being positioned between the amino acid sequence of HBP and the amino acid sequence of the second polypeptide, wherein the second polypeptide provides the fusion protein with capabilities of forming insoluble aggregates in the cytoplasm of bacteria after being expressed in said bacteria.
  • a polypeptide sequence of HBP is positioned C-terminally in the fusion protein, and a second polypeptide sequence is positioned N-terminally.
  • the second polypeptide of the fusion protein may be a homologous polypeptide, such as defined above, or a heterologous polypeptide.
  • heterologous polypeptide is meant a polypeptide with the amino acid sequence which has at maximum about an 80 % identity with the amino acid sequence of HPB set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, and 17.
  • said heterologous polypeptide may be a calf chymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptide fragments thereof.
  • the heterologous polypeptide is a bacterial thioredoxin or human, natural or synthetic variants, or peptide fragments thereof.
  • the heterologous polypeptide is human ubiquitin, natural or synthetic variants, or peptide fragments thereof.
  • the heterologous polypeptide is fused in frame to HBP through a polypeptide sequence comprising a protease cleavage site.
  • the protease cleavage site may be a Factor Xa, with the amino acid sequence IEGR, enterokinase, with the amino acid sequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, or Achromobacter lyticus, with the amino acid sequence K, cleavage site.
  • the protease cleavage site is a Achromobacter lyticus cleavage site.
  • a recombinant vector including the DNA construct encoding a fusion protein, wherein said fusion protein comprises HBP, a second polypeptide and a protease cleavage site is used to transform the host cell to express the fusion protein.
  • a useful host cell may be a cell of bacteria such as gram positive bacteria including, but not limited to, a Bacillus cell, e. g. Bacillus alkalophilus, Bacillus amyloliquefa- ciens, Bacillus brevis, Bacillus cieculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e. g. Streptomy- ces lividans or Streptomyces murinus, or gram negative bacteria such as E.
  • Bacillus cell e. g. Bacillus alkalophilus, Bacillus amyloliquefa- ciens, Bacillus brevis, Bacillus cieculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,
  • the bacterial host cell is selected from the group conprising the cells of Bacillus subtilis, Bacillus brevis and E. coli. In a more preferred embodiment, the bacterial host cell is a E. coli cell.
  • the transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using complement cells (Young and Spizizin, 1961 , J. Bacteriol. 81 :823-829; Dubnau and Davidoff Abelson, 1971, J. Mol. Biol. 56:209-221), electro- poration (Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (Koehler and Thome, 1987, J. Bacteriol. 169:5771-5278).
  • the transformed cells are further cultured in any conventional medium suitable for growing prokaryotic cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e. g. in catalogues of American Type Cul- ture Collection). The cells are then screened for antibiotic resistance. Subsequently, the selected clones are assayed for HBP activity using assays known in the art such as a chemotaxis assay and testing cytokine release from monocytes (see, for example US 5,814,602).
  • assays known in the art such as a chemotaxis assay and testing cytokine release from monocytes (see, for example US 5,814,602).
  • the fusion protein When expressed in bacteria such as E. coli, the fusion protein may retain in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be di- rected to the periplasmatic space by bacterial secretion sequence. It is a further aspect of the present invention to provide a method for simple, but effective isolation and purification of recombinant HBP by using the advantage of a surprising expression of the HBP-fusion protein in bacteria in insoluble form and accumulation of said protein in inclusion bodies in the cytoplasm of bacteria.
  • Inclusion bodies containing the fusion protein of the invention is to be isolated by a one-step centrifugation of the crude cell lysate.
  • the fusion protein accumulated in the inclusion bodies is dissolved in aqueous solution containing a detergent, e. g. guanidine hydrochloride, urea or sarkosyl.
  • a detergent e. g. guanidine hydrochloride, urea or sarkosyl.
  • a reducing agent is added to break intra and inter molecular disulphide bonds. The latter is important for subsequent refolding of the protein.
  • the fusion protein may be advantageously cleaved with a suitable protease to isolate HBP from a fusion partner.
  • the suitable protease is selected according to the protease cleavage site within the fusion protein.
  • a suitable protease may be Factor Xa, enterokinase, thrombin, or the Achromobacter lyticus protease.
  • a suitable protease is the Achromobacter lyticus protease.
  • Protein refolding may be a unique series of opera- tions that involves isolation of the protein of interest, dissolving the protein in strong denaturants, preparing the dissolved protein for refolding and then recovering the biological activity by controlled removal of the denaturant (refolding procedure).
  • the protein must be refolded to the native conformation.
  • a major obstacle to achieving high refolding yields is the propensity of the solubilised, unfolded protein to form irreversible aggregates rather than to proceed to the fully folded native state.
  • various techniques are known in the art to overcome this problem, the approach to the protein refolding is unique for every protein.
  • the most commonly used techniques include but are not limited to factorial design refolding focused on dilution and re-oxidation of the protein, refolding by denaturant removal and re-oxidation using dialysis (a conventional dialysis as well as the hollow-fibre dialysis), refolding by interactive denaturant (addition and removal), refolding by use of a molecular chaperone (GroEL), artificial chaperone-assisted refolding, micelle-assisted refolding and co-solvent-assisted refolding.
  • GaEL molecular chaperone
  • recombinant HBP is refolded by size-exclusion chromatography and equilibrated with refolding buffer containing a reducing/oxidizing system.
  • refolding procedure of WO 94 18227 (Holtet et al.) is used for refolding HBP.
  • the folded recombinant HBP may be used as an active component for the preparation of a pharmaceutical antimicrobal composition for treatment of a mammal having a bacterial disease state.
  • Fig. 1 illustrates the components of two expression plasmids for mature HBP and precursor HBP, pHW1280 and pHW1283, respectively.
  • Most of the coding region of HBP is taken out as a 700 bp Eag1-Xho1 fragment from pSX558, harbouring the coding region of HBP as described in Almeida et al., 1991, Biochem. Biophys. Res.Comm. 177:688-95.
  • This fragment is ligated to 4.2 kb Nco1-Xho1 fragment of the Invitrogen expression vector pSE380 and the linker 4813/4814 for mature HBP or the linker 4801/4802 for pro-HBP:
  • pHW1280 and pHW1283 were transformed into E. coli TOP10 (Invitrogen) and cells were grown in LB medium at 37 °C. At OD 450 around 1.0, cells were induced with 1 mM IPTG for 3 h and whole cell lysates were analysed by SDS-PAGE.
  • Fig. 7a shows proteins expressed in cells transformed with pHW1280 (lanes 2 and 3) and pHW1283 (lanes 5 and 6). Neither mature, nor pro-HBP appeared as new protein bands after induction. The mature HBP was also inserted into the expression vector pSE280, yielding the plasmid pHW1282.
  • Induction of the expression from pSE380 is strictly controlled by lacP on the plasmid, while pSE280 has more relaxed control, by the one-copy lacl on the chromosome. However, using pSE280 did not improve the yield of recombinant HBP.
  • Example 2 Cytoplasmic expression of HBP fused to various lengths of calf chymosin.
  • Fusion to other proteins is often used to provide a purification tag for expressed protein and/or to increase expression yield by adding N-terminally a well expressed protein to act as a 'locomotive' for the synthesis.
  • Another reason for fusing proteins for expression could be that the fusion partner might shield the protein of interest from degradation by proteases, or the complex might obtain solubility properties different from the fusion partners. In the case of HBP, considering the toxicity of the protein to E.coli, a decreased solubility would seem preferable.
  • pHW1311 the wildtype sequence of prochymosin, as stated in Harris et al. Nucleic Acids Res. 10, 2177 (1982), was used.
  • pHW1312 the cysteines in positions 98 and 103 were mutated to serines and the lysines in positions 99 and 104 were mutated to arginines to avoid possible interference form the prochymosin part during processing, in the formation of disulphide bridges and in the cleavage of the fusion protein.
  • the two plasmids were transformed into E.coli K12 W3110 Iq and propagated as described in Examplel .
  • Glutathione S-transferase from S.japonicum was chosen at random as a fu- sion partner in an attempt to increase the yield and survival of HBP in E.coli.
  • the plasmid pGEX-2T (Pharmacia) with a thrombin cleavage site was chosen for construction of the GST-pro-HBP fusion protein.
  • This vector was cut into two fragments, BamH1-AlwN1 of 3.0 kb and AlwN1-Sma1 of 1.9 kb. They were ligated to the 700 bp Eag1-EcoR5 HBP gene and to the 4825/4826 BamH1-Eag1 linker to yield the expression plasmid pHW1296 :
  • the expression plasmids for thioredoxin fused to mature and pro-HBP are outlined in Fig. 4, all having the enterokinase site for cleavage. Common elements are the 700 bp Eag 1-Xho 1 HBP fragment of pSX558 and two fragments, AlwN 1-Crf 101 1.6 kb and AlwN 1-Sal 1 2.0 kb, of pTrxFus (Invitrogen).
  • the mature HBP is in two versions, with (pHWl288) and without (pHW1289) methionine at the N-terminus of the cleaved HBP, and with the NgoM1-Eag1 linkers 4839/4840 and 4837/4838, respectively:
  • the three plasmids, pHW1288, 1289 and 1290, were transformed into E. coli Gl 724 competent cells (Invitrogene) harbouring the ⁇ cl repressor gene on the chromosome for regulated expression of thioredoxin fusions from ⁇ P L promotor.
  • the repressor is under control of the trp promotor , so induction is initiated by addition of tryptophan 100 ⁇ g/ml, which prevents further synthesis of repressor, allowing the ⁇ P L promoter to work and fusion proteins to be expressed.
  • Cells are grown in tryptophan depleted RM medium before induction.
  • Fig. 7 shows the fusion protein expression in the transformants of pHW 1288 (7a, lanes 7, 8 and 9), pHW1289 (7a, lanes 10, 11 and 12) and pHW1290 (7a, lanes 13, 14 and
  • the yield is estimated to be about 50 mg/L/OD 450 in all 3 constructs.
  • Example 5 Expression of HBP, wild type and the K6R mutant, fused to thioredoxin via an Achromobacter lyticus protease A cleavage site in E. coli
  • Protease A from the bacterium Achromobacter lyticus is a very robust enzyme with properties required for a good production process. It is a strictly lysine specific enzyme, with no tendency to cleave at arginine, and the protein to be produced must therefore be devoid of lysines or have any lysines well protected from cleavage.
  • Human HBP has only one lysine at position 6 in the mature protein, and since the porcine variant of HBP has arginine in this position, the mutation K6R may not have a significant influence on the protein function.
  • the PCR primers are used to introduce the mutation: 5177: 5' ACATCGTTGGCGGCCGGCGTGCGAGGCC 3' (SEQ ID NO: 37) AAG>CGT
  • Primer 5177 has the upstream Eag 1 restriction site, which was also used in the other examples, and the primer 5178 has a Sac 2 site 100 bp downstream of Eag 1.
  • the 100 bp Eag 1-Sac 2 fragment was ligated to the sequence of 230 bp between Sac 2 and Pst 1 of the HBP coding sequence, and the following Pst 1- Eag 1 fragment of 3.1 kb of pSx555 harboring the HBP gene itself, as seen in Fig. 5, resulting in the plasmid pHW1302.
  • the enterokinase proteolytic site is altered to an A. lyticus protease site by exchanging the NgoM l-Eag 1 linker in pHW1289 with the 4B ALP/4B ALP2 linker:
  • ALP2 3' GAGACCAAGACCACGTCGGGGCTTTTAGCACCCGCCGG 5' (SEQ ID NO: 40)
  • a modified ubiquitin gene His-tagged with KH8 at the N-terminal and with a Sac2 site introduced near the C-terminal was inserted in a pET derived vector pHW 1376, as shown in Fig.6, in which the inserted cytokine gene was substituted with the 700 bp Eag1 - Xho1 fragment covering the HBP gene, connecting the two with the FXa- site cleavage linker:
  • the resulting expression plasmid pHW1380 primarily established in E.coli MC1061 was unable to express the protein from the T7 promoter, and after verification it was further transformed into E.coli BL21 DE3 harbouring the T7 polymerase in the genome.
  • Fig. 6 also illustrates the construction of pHW1383, which has the mutated HBP
  • the yield of the recombinant protein was estimated to be about 100 mg/L/OD450.
  • Expression of the HBP-ubiquitin fusion proteins is shown on Fig. 7b, lanes 14, 15 and 16 (pHW1380) and 7b, lanes 17, 18 and 19 (pHW1383).
  • Example 7 Preparation of recombinant HBP from inclusion bodies. By the following preparation the expressed HBP-fusion may be recovered from inclusion bodies. Composition of the buffers: Buffer A: 50 mM tris(hydroxymethyl)aminonethane (Tris),
  • E. coli cell pellet from 1 I culture flask is resuspended in 75 ml ice-cold lysis buffer and incubated with 0.37 ml of a lysozyme solution (10 mg lysozyme/ml buffer A) for 20 min on ice.
  • the suspension is sonicated 40Wx10 sec (9 power impulses with 10 sec silence intervals).
  • the sonicated sample is centrifuged 10,000 gx10 min at 4 °C.
  • the pellet is resuspended in 75 ml buffer C and centrifuged as before.
  • the pellet is next suspended and incubated in 60 ml extraction buffer for 1 ,5 h at room temperature with vigorous shaking.
  • the resulting extract is centrifuged as before.
  • a sample of supernatant is analysed by SDS-PAGE for detection of the extracted HBP-fusion protein.
  • the supernatant is further diluted with buffer G (1 :1) and digested with a protease according to the protease cleavage site expressed in the fusion protein.
  • the enzyme is applied in a dilution of 1:10-1 :100 adjusted according to the protein concentration in the inclusion bodies extract. Digestion is performed at room temperature for 2-18 h.
  • the digested sample is first purified on the G-25 Sephadex gel filtration column equilibrated with buffer E, and next on the CM-Sepharose ion exchange column using a gradient of concentra- tion of NaCl (buffer F). Purified samples are analysed by SDS-PAGE, HPLC, capillary elecfrophoresis, N-terminal sequence analysis and mass-spectrometry
  • the column is run with flow rates 0.1-1 ml/min at 25 °C or 4 °C.
  • the experiments are performed using an Akta explorer system. Fractions are collected and analysed by HPLC. Selected fractions containing putative refolded protein are tested for biological activity in a SPA based aprotinin and/or LPS binding assay, and monocyte activation assay.
  • a solution of purified HBP as described in Example 6 is converted from buffer F to a guanidinium hydrochloride buffer (e. g. 10-50 mM Tris sulphate, 3-6 M guanidinium hydrochloride, 2-10 mM DTT, 2 mM EDTA, pH 7-8.5) or an urea buffer (e. g. 50 mM Tris sulphate, 4-6 M urea, 2-10 M DTT, 2 mM EDTA, pH 7-8.5) by chromatography on Sephadex G-25 followed by ultrafiltration to concentrate the sample, or by successive repetitive ultrafiltrations alone for both buffer conversion, and concentrating the sample.
  • a guanidinium hydrochloride buffer e. g. 10-50 mM Tris sulphate, 3-6 M guanidinium hydrochloride, 2-10 mM DTT, 2 mM EDTA, pH 7-8.
  • an urea buffer e. g. 50 mM Tris
  • a sample of HBP in one of the above buffers is diluted to 10-500 ⁇ g protein per ml and concentration of DTT and EDTA is reduced to 0.1-0.5 mM and to 1 mM correspondingly, and at the same time the glutathion redox system (reduced/oxidised glutathione ratio being 1 :2-1 :10) and an artificial chaperone (detergent) at concentration over the critical micelle limit (e. g. at least
  • Table 1 shows the relevant contents of the plasmids used in the examples above: pHW 1280 met HBP pHW 1283 met pro Entero HBP
  • FXa IEGR / Thrombin : LVPR / GS Enterokinase : DDDDK / ALP : A.lyticus protease : K /

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Abstract

L'invention concerne des procédés de préparation d'une protéine de liaison à l'héparine (HBP) dans un système d'expression bactérien recombiné. Plus particulièrement, l'invention concerne un procédé de préparation d'une protéine de fusion insoluble comprenant une protéine de liaison à l'héparine (HBP), un site de clivage protéolytique, et un second polypeptide dans des cellules bactériennes recombinées, ladite protéine de fusion étant accumulée dans des corps d'inclusion siégeant dans le cytoplasme d'une bactérie après l'expression. Par ailleurs, l'invention concerne des procédés de séparation de la HBP exprimée de corps d'inclusion qui consistent à purifier la HBP du second polypeptide, et éventuellement, replier ladite HBP. De plus, l'invention concerne des produits de recombinaison d'ADN qui comprennent différentes protéines de fusion HBP. L'invention concerne enfin l'utilisation de la protéine de fusion HBP d'origine bactérienne dans la production de HBP pure, ainsi que l'utilisation de la HBP purifiée d'origine bactérienne dans la préparation d'un médicament.
PCT/DK2003/000207 2002-03-27 2003-03-26 Procede de preparation d'une proteine de liaison a l'heparine (hbp) mammifere recombinee WO2003080660A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013019604A3 (fr) * 2011-07-29 2013-04-25 Georgia State University Research Foundation, Inc. Procédé de production de protéines
CN117343200A (zh) * 2023-12-04 2024-01-05 北京质肽生物医药科技有限公司 一种包含淀粉样核心多肽的融合蛋白、制备方法及其应用
CN117965667A (zh) * 2024-01-23 2024-05-03 陕西普罗安蒂生物科技发展有限公司 一种通过切割融合蛋白制备多肽的方法

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CN108018294B (zh) * 2018-01-02 2020-11-17 昆明理工大学 一种三七植物激素结合蛋白基因PnPhBP1及应用

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HU219883B (hu) * 1988-03-17 2001-08-28 Novo-Nordisk A/S Heparinkötő proteinek, ezeket kódoló DNS és a proteineket tartalmazó gyógyszerkészítmények, valamint eljárás ezek előállítására
US5484885A (en) * 1989-07-05 1996-01-16 Emory University Chemotactic, antibiotic and lipopolysaccharide-binding peptide fragments of CAP37
WO1999000417A1 (fr) * 1997-06-25 1999-01-07 Novo Nordisk A/S Procede d'obtention de proteines fixant l'heparine dans des cellules de mammiferes
EP1032412A1 (fr) * 1997-11-20 2000-09-06 Novo Nordisk A/S Utilisation de proteine de liaison a l'heparine pour la modulation ou la prophylaxie de l'apoptose des cellules de mammiferes
KR20020034073A (ko) * 1999-04-29 2002-05-08 레우코테크 에이/에스 재조합 포유동물 세포에서 헤파린-결합 단백질의 발현

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013019604A3 (fr) * 2011-07-29 2013-04-25 Georgia State University Research Foundation, Inc. Procédé de production de protéines
CN117343200A (zh) * 2023-12-04 2024-01-05 北京质肽生物医药科技有限公司 一种包含淀粉样核心多肽的融合蛋白、制备方法及其应用
CN117343200B (zh) * 2023-12-04 2024-01-30 北京质肽生物医药科技有限公司 一种包含淀粉样核心多肽的融合蛋白、制备方法及其应用
CN117965667A (zh) * 2024-01-23 2024-05-03 陕西普罗安蒂生物科技发展有限公司 一种通过切割融合蛋白制备多肽的方法

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