WO2003080660A2

WO2003080660A2 - Method for the preparation of recombinant mammalian heparin-binding protein (hbp)

Info

Publication number: WO2003080660A2
Application number: PCT/DK2003/000207
Authority: WO
Inventors: Helle Fabricius WÖLDIKE
Original assignee: Leukotech AS
Current assignee: Leukotech AS
Priority date: 2002-03-27
Filing date: 2003-03-26
Publication date: 2003-10-02
Anticipated expiration: 2004-09-27
Also published as: WO2003080660A3; AU2003226907A1; AU2003226907A8

Abstract

The present invention relates to methods of making heparin-binding protein (HBP) in a recombinant bacterial expression system. In particular, this invention relates to a method for the preparation of an insoluble fusion protein comprising heparin-binding protein (HBP), a proteolytic cleavage- site, and a second polypeptide in recombinant bacterial cells, said fusion protein being accumulated in inclusion bodies in the cytoplasm of bacterium after expression. The invention further relates to methods of separation of the expressed HBP from inclusion bodies comprising purification of HBP from the second polypeptide and optionally refolding of said HBP. The invention further features the DNA constructs comprising different HBP-fusion proteins. Invention also relates to use of the HBP-fusion protein produced in bacteria for the production of pure HBP and use of the HBP purified from the fusion protein for the preparation of a medicament.

Description

Method for the preparation of recombinant mammalian heparin-binding protein (HBP)

Field of the invention

The present invention relates to methods of making heparin-binding protein (HBP) in a recombinant bacterial expression system. In particular, 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.

Throughout this application, various publications are referenced. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more detailed describe the state of the art to which this invention pertains.

Background of the invention

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. In the process of inflammation 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. 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. The protein has been named human heparin-binding protein (hHBP), owing to its high affinity for heparin binding. A highly homologous protein was also isolated from PMNs of porcine origin and has been named porcine heparin-binding protein (pHBP) correspondingly. (H. Flodgaard et al., 1991, Eur. J. Biochem. 197: 535-547; J. Pohl et al., 1990, FEBS Lett. 272: 200 ff.) HBP was originally studied because of its antibiotic and lipopolysaccharide binding properties (Gabay et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:5610-5614 and Pereira et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4733-7). However, a number of experimental evidence now supports the concept that HBP, in addition to its bacteri- cidal role, is involved during the progression of inflammation due to its effect on the recruitment and activation of monocytes (Pereira et al., 1990, J. Clin. Invest. 85:1468-1476, and Rasmussen et al., 1996, FEBS Lett. 390:109-112), recruitment of T cells (Chertov et al., 1996, J. Biol. Chem. 271 :2935-2940), as well as on the induced contraction in endothelial cells and fibroblasts (Ostergaard and Flodgaard, 1992, J. Leuk. Biol. 51 :316-323). Ostergaard and Flodgaard, 1992, op. cit. also disclose increased survival of monocytes treated with heparin-binding protein. Furthermore, in animal models of fecal peritonitis, HBP treatment has been shown to rescue mice from an otherwise lethal injury (Mercer-Jones et al., 1996, In Surgical Forum, pp. 105-108 and Wickel et al., 1997, In 4th International Congress on the Immune Consequences of Trauma, Chock and Sepsis, Munich, Germany, pp. 413-

416).

The structure of HBP appears from WO 89/08666 and H. Flodgaard et al., op. cit. 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).

Production of HBP in recombinant bacterial cells has been disclosed in US

5,484,885. 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). Using this system expressed 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-

GST in recombinant bacteria, nor the yield of the expressed protein.

It would be advantageous to produce an anti-microbial recombinant protein in a cell of bacterium in precipitated form, as (1) the precipitated protein is protected from degradation by host proteases, (2) the inert insoluble protein is unable to seriously harm bacterium, (3) the protein precipitation gives a better yield of expression, and (4) precipitation of a protein in inclusion bodies simplifies purification of the protein allowing a one-step purification of said protein from the bulk of soluble bacterial proteins by centifugation of bacterial cell lysate.

Thus, it is an objective of the invention to obtain recombinant heparin-binding protein in high yields in a simple but efficient manner by using a bacterial expression system.

Summary of invention

It has surprisingly been found that heparin-binding protein (HBP), when fused to a polypeptide that in the majority of cases increases solubility of recombinant proteins expressed in bacteria, is accumulated in insoluble inclusion bodies in the cytoplasm of recombinant bacterial cells. It is advantageous to produce recombinant HBP in a cell of bacterium in precipitated form, as (1) the precipitated fusion protein is protected from degradation by host proteases, (2) the inert insoluble protein is unable to seriously harm bacterium, (3) the protein precipitation gives the better yield of expression, and (4) precipitation of a fusion protein in inclusion bodies simplifies purifi- cation of the protein allowing a one-step purification of said protein from the bulk of soluble bacterial proteins by centifugation of bacterial cell lysate.

In one aspect 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;

b) transforming host cells of bacterium with the recombinant vector of step (a);

c) culturing the transformed host cells of step (b) which express and accumulate the fusion protein of step (a) in insoluble form in the cytoplasm;

d) lysing the cells of step (c);

e) obtaining a precipitate comprising the fusion protein in the insoluble fraction of the host cell lysate of step (d).

In another aspect the invention provides a method for producing of recombinant heparin-binding protein (HBP) in 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;

d) lysing the cells of step (c);

e) obtaining the expressed fusion protein in the insoluble fraction of the host cell lysate of step (d);

f) dissolving the obtained fusion protein of step (e) in aqueous solution;

g) cleaving the solved fusion protein of step (f); h) purifying HBP after the cleavage of step (g), and optionally refolding HBP obtained after purification.

In yet another aspect the invention provides a method for producing recombinant heparin-binding protein (HBP) in bacterial cells comprising,

a) providing an insoluble form of a fusion protein, wherein said fusion protein comprises HBP, a second polypeptide and a protease cleavage site,

b) dissolving the fusion protein in aqueous solution;

c) cleaving the solved fusion protein of step (b);

d) purifying HBP after the cleavage of step (c);

e) optionally refolding HBP obtained after purification of step (d).

In still yet another aspect the invention provides a method for producing in bacterial cells a mammalian heparin-binding protein (HBP) comprising,

a) culturing a recombinant bacterial cell transformed by an expression vector including a hybrid gene comprising a nucleic acid sequence encoding HBP, which is is fused in frame to a sequence encoding a protease cleavage site, which in turn is fused in frame to a sequence encoding a second polypeptide in a suitable culture medium under conditions permitting expression and accumulation of said fusion protein in a form of inclusion body in the cytoplasm of said cells;

b) isolating the inclusion body of (a);

c) dissolving said inclusion body in an aqueous solution;

d) cleaving the dissolved fusion protein of (c);

f) purifying HBP after the cleavage of (d); e) optionally refolding the purified HBP of (f).

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.

Advantages accompanying the use of the method and constructs described herein include the fact that the separation of the fusion protein from the bulk of bacterial proteins is a simple, one-step centrifugation and that the expressed fusion protein is a stable protein and can be recovered in high yield. These improvements provide a further feature of the invention, which resides in the use of the fusion protein for producing a heparin-binding protein (HBP), and a yet further feature of the invention, which resides in the use of the heparin-binding protein (HBP) for the preparation of a medicament.

Description of Drawings Fig. 1 Outline of construction of the expression plasmids pHW1280 and pHW1283 for the mature human HBP and precursor human HBP, pHW1280 and pHW1283, respectively (Example 1 ).

Fig. 2 Outline of construction of the expression plasmids pHW1311 and pHW1312 encoding the mature human HBP fused to the wild type and mutated calf prochymosin respectively (see details in Example 2).

Fig. 3 Outline of construction of the expression plasmids pHW1295 and pHW1296: pHW1295 encodes the mature human HBP fused to glutathione-S-reductase (GST) via the factor Xa protease cleavage site, and 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,

18); b- pHW1295 (lanes 1, 2, 3), pHWl296 (lanes 4, 5, 6), pHW1311 (lanes 7, 8, 9),

PHW1312 (lanes 10, 11, 12), pHW1380 (lanes 14, 15, 16), pHW1383 (lanes

17, 18, 19), separated by SDS-PAGE and stained with Coomassie. 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 ).

Detailed description of the invention

HEPARIN-BINDING PROTEIN (HBP)

In the present context by HBP is meant 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,

In natural form 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.

According to the invention, 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.

It is an objective of the invention to obtain recombinant HBP having the activities as defined above in high yields in a simple, but efficient manner by using a bacterial expression system. Thus, in one aspect 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. In particular, 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"). Alternatively, 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)

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.

In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by at most five amino acids, preferably by at most four amino acids, more preferably by at most three amino acids, even more preferably by at most two amino acids, and most preferably by at most one amino acid from the amino acid sequence set forth in SEQ ID NOS:1 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 pack- age (Needleman and Wunsch, 1970, J. Mol. Biol. 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. The 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. Preferably, 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.

Examples of 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

York. The most commonly occurring exchanges are: Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, Asp/Gly as well as these in reverse.

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: 11), SEQ ID NO: 14 (which encodes a porcine HBP which includes the pro sequence and sequence of the mature protein, depicted in SEQ ID NO:13), SEQ ID NO: 16 (which encodes a porcine HBP which includes the signal sequence, pro sequence and sequence of the mature protein, depicted in SEQ ID NO:15), SEQ ID NO:18 (which encodes a porcine HBP which includes the signal sequence and sequence of the mature protein, depicted in SEQ ID NO: 17), more preferably at least about 70% identity, more preferably at least about 80% identity more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%. The nucleic acid sequence may be of genomic, cDNA, 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, 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.

Modification of the nucleic acid sequence encoding HBP may be necessary for the synthesis of polypeptide sequences substantially similar to HBP. The term "substantially 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 synthesise variants of HBP where the variants differ in specific activity, thermostability, pH op- timum, 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: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. For a general description of nucleotide substitution, see, e.g., 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 can be made outside the regions critical to the function of the molecule and still result in an active polypeptide sequence. Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, 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 recombinant heparin-binding protein may also be encoded by a nucleic acid sequence that hybridizes to a nucleic acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16 and 18 at low to high stringency conditions. Low to high strin- gency conditions are defined as pre-hybridization and hybridization at 42°C in 5X

SSPE, 0.3% SDS, 200 ug/ml sheared and denatured salmon sperm DNA and either 25, 35 or 50% formamide for low, medium and high stringencies, respectively. 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).

CONSTRUCTION AND EXPRESSION OF THE HBP-FUSION PROTEIN The fusion protein: DNA construct and recombinant expression vector

According to the present invention, 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.

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. In one embodiment, 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. In a more preferred embodiment, 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. In an even more preferred embodiment, the DNA sequence encoding a second polypeptide is a sequence, which hybridises to the DNA sequence of ubiquitin. In another embodiment, 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.

In the nucleic sequence of the invention the DNA sequences encoding partner polypeptides of the fusion protein are fused in frame giving a DNA construct encoding the fusion protein. In the preferred embodiment 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.

The 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. Thus, 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). Alternatively, 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.

In the vector, the 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. Examples of 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. coli lac operon, the T5 and T7 bacteriophages, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus lichenifor- mis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amy- lase gene (amyM), the Bacillus amyloliquefaciens alpha amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Pro- ceedings of the National Academy of Sciences USA 75:3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Nat. Acad. Sci. USA 80:21 25). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989. The nucleic acid sequence encoding the fusion protein of the invention may be op- erably connected to a suitable terminator of transcription.

In the preferred embodiment, 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. When the host cell is a bacterial cell, 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.

The procedures used to ligate the nucleic acid sequences coding for the fusion protein, the promotor and terminator, respectively, and to insert them into suitable vec- tors containing the information necessary for replication, are well known to persons skilled in the art (Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989).

The fusion protein: a fusion partner polypeptide and cleavage site

According to the invention, 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. In the preferred embodiment, 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. In the present content by "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. In one embodiment, said heterologous polypeptide may be a calf chymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptide fragments thereof. In a more preferred embodiment, the heterologous polypeptide is a bacterial thioredoxin or human, natural or synthetic variants, or peptide fragments thereof. In the most pre- ferred embodiment, 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. In one embodiment, 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. In a preferred embodiment, the protease cleavage site is a Achromobacter lyticus cleavage site.

Expression of the fusion protein: a host cell

According to the method of the invention, 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. coli or Pseudomonas sp. In a preferred embodiment, 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).

Isolation of the fusion protein and use of said protein for production of recombinant HBP

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.

Advantage accompanies the "one-step" isolation of the inclusion bodies is also pro- vided by a high recovery level of the fusion protein, and the latter feature of the method of the invention provides an additional aspect of the invention, which resides in use of the recovered from inclusion bodies fusion protein for production of a biologically active mammalian HBP.

The fusion protein accumulated in the inclusion bodies is dissolved in aqueous solution containing 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.

After recovery from inclusion bodies and before refolding, 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. In one embodiment, a suitable protease may be Factor Xa, enterokinase, thrombin, or the Achromobacter lyticus protease. In a preferred em- bodiment, a suitable protease is the Achromobacter lyticus protease.

Before refolding the recombinant HBP protein, it might be necessary to further purify said protein, since the contaminating polypeptide left from the digested fusion protein can interfere with the refolding process. Ion exchange, hydrophobic interaction and gel filtration chromatography can be performed using a urea dissolved protein.

Working with a guanidine hydrochloride dissolved protein sample purification is limited to gel filtration, ultra filtration and dialysis.

HBP is subsequently refolded. 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. Although 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. In one preferred embodiment, recombinant HBP is refolded by size-exclusion chromatography and equilibrated with refolding buffer containing a reducing/oxidizing system. In a more preferred embodiment, the refolding procedure of WO 94 18227 (Holtet et al.) is used for refolding HBP.

It is yet another aspect of the invention to use the produced as defined above HBP for the preparation of a medicament.

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.

Examples

Example 1. Cytoplasmic expression of pro- and mature HBP in E. coli

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:

4813: 5' CATGATCGTCGGC 3' (SEQ ID NO: 19) 4814: 3' TAGCAGCCGCCGG 5' (SEQ ID NO: 20)

4801 :

5' CATGGGCAGCAGCCCGCTGCTGGATGACGATGACAAAATCGTCGGC

(SEQ ID NO: 21)

4802: 3' CCGTCGTCGGGCGACGACCTACTGCTACTGTTTTAGCAGCCGCCGG 5'

(SEQ ID NO: 22)

pHW1280 and pHW1283 were transformed into E. coli TOP10 (Invitrogen) and cells were grown in LB medium at 37 °C. At OD₄₅₀ 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.

Full length or parts of calf prochymosin when synthesized in the cytoplasm in E.coli is precipitated in inclusion bodies. A series of fusion proteins of HBP to various lengths of prochymosin was produced with expectation of a good yield of precipitated fusion protein. One of the constructs is outlined in Fig.2. From pHW261 har- bouring prochymosin fused N-terminally to MTMITNSAA from pUC7, a fusion is made from Sma1 in prochymosin corresponding to amino acid 161 in the extended protein, to Eag1 in the HBP gene, connecting the two with the 'Sma1'-Eag1 linker:

5173 : 5' AAGATCGTTGGC 3' (SEQ ID NO: 23) 5174 : 3' TTCTAGCAACCGCCGG 5'(SEQ ID NO: 24) In pHW1311 the wildtype sequence of prochymosin, as stated in Harris et al. Nucleic Acids Res. 10, 2177 (1982), was used. In 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 .

As seen in FigJb, lanes 7-12, the yield of a fusion protein (of about 43 kD) expressed from the two above constructs turned out to be modest.

Example 3. Cytoplasmic expression of GST-HBP fusion proteins in E. coli

Glutathione S-transferase (GST) 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 construction of expression plasmids for mature and pro-HBP fused to GST is outlined in Fig.3. In both cases pSX555 donates the HBP gene as a 700 bp Eag1- EcoR5 fragment. For the mature HBP this is ligated to pGEX-5X-3 (Pharmacia), harbouring a Factor Xa cleavage site, and to the 4815/4816 'BamH1'-Eag1 linker to yield the expression plasmid pHW1295 :

4815 : 5' GATCGTGGGC 3' (SEQ ID NO: 25)

4816 : 3' CACCCGCCGG 5' (SEQ ID NO: 26)

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 :

4825 : 5' GATCCAGCCCGCTGCTGGATATCGTGGGC 3' (SEQ ID NO: 27) 4826 : 3' GTCGGGCGACGACCTATAGCACCCGCCGG 5' (SEQ ID NO: 28) pHW1295 and 1296 were transformed into E.coli MC1061 (Wertman, K.F. et al. (1986) Gene 49:253-262). The transformants were tested for protein expression as described in Example 1. Fig. 7b demonstrates proteins from cell extracts before and after 3h induction of protein expression with IPTG separated by SDS-PAGE and stained with Coomassie. Distinct, new protein bands of expected size around 50 kD, are seen after 3 h induction of expression of both mature and pro-HBP fused to GST (Fig. 7b, lanes 2 and 3). The yield of the expression is estimated to be about 30 mg/L/OD450. Inspection of cell extracts in the microscope reveals precipitation of the fusion protein in inclusion bodies.

Example 4. Expression of thioredoxin-HBP fusion proteins in E. coli

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:

4839: 5' CCGGCTCTGGTTCTGGTGATGACGATGACAAAATGATCGTGGGC 3' (SEQ ID NO: 29) 4840: 3' GAGACCAAGACCACTACTGCTACTGTTTTACTAGCACCCGCCGG 5' (SEQ ID NO: 30)

4837: 5' CCGGCTCTGGTTCTGGTGATGACGATGACAAAATCGTGGGC 3' (SEQ ID NO: 31) 4838: 3' GAGACCAAGACCACTACTGCTACTGTTTTAGCACCCGCCGG 5' (SEQ ID NO: 32)

For the expression plasmid pHW1290 for pro-HBP, the NgoM1-Eag1 linker 4829/4845 was used: 4829:

5' CCGGCTCTGGTTCTGGTGATGACGATGACAAAGGCAGCAGCCCGCTGC 3'

(SEQ ID NO: 33)

4845:

3' AGACCAAGACCACTACTGCTACTGTTTCCGCCGTCGTCGGGCGACGACCTA 5'

(SEQ ID NO: 34)

4829: 5' TGGATATCGTGGGC 3' (SEQ ID NO: 35) 4845: 3' TAGCACCCGCCGG 5' (SEQ ID NO: 36)

The three plasmids, pHW1288, 1289 and 1290, were transformed into E. coli Gl 724 competent cells (Invitrogene) harbouring the λcl repressor gene on the chromosome for regulated expression of thioredoxin fusions from λP_L promotor. The repressor is under control of the trp promotor , so induction is initiated by addition of tryptophan 100 μg/ml, which prevents further synthesis of repressor, allowing the λP_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

15) before and after 3 h induction with tryptophan. The yield is estimated to be about 50 mg/L/OD₄₅₀ 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 (A. 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

5178: 5' AAGCAGCTGGCCGCGGTCATCACG 3' (SEQ ID NO: 38)

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. After initial cloning into the TA vector (Invitrogen), 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.

In the expression plasmid pHW1289 encoding the thioredoxin-HBP fusion protein, 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:

4B ALP1 : 5' CCGGCTCTGGTTCTGGTGCAGCCCCGAAAATCGTGGGC 3' (SEQ ID NO: 39)

4B ALP2: 3' GAGACCAAGACCACGTCGGGGCTTTTAGCACCCGCCGG 5' (SEQ ID NO: 40)

The linker introduces a short spacer between thioredoxin and HBP in the fusion protein with the amino acid sequence GSGSGAAPK, with alanine and proline being some of the preferred amino acids in front the lysine residue for efficient cleavage. The resulting plasmid pHW1304 (Fig. 5) is cut by Eag 1 and BspE 1 endonucleases into two fragments, of 700 bp and 3.0 kb, and ligated into the pHW1302 Eag 1-Dra 3 fragment of 550 bp, thus bringing together the A. lyticus protease site and the K6R mutation in the expression plasmid pHW1306. The fusion protein expression yield of E. coli Gl 724 clones transformed with pHW1306 encoding the mutated human HBP (Fig. 7a, lanes 16, 17 and 18) is comparable in yield to the pHW1289 transformants expressing the wild type human HBP (Fig. 7a, lanes 11, 12 and 13). Example 6. Cytoplasmic expression of the ubiquitin-HBP fusion protein in E. coli

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:

Sac2 - Eag1 FXa :

5' GGTGGTATCGAAGGCCGTATCGTGGGC 3' (SEQ ID NO: 41)

3' CGCCACCATAGCTTCCGGCATAGCACCCGCCGG 5' (SEQ ID NO: 42)

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

K6R and a linker providing the A. lyticus protease site:

Sac2-Eag1 ALP:

5' GGGTGGCAGCCCGGTAAGCGGTGCGGCGCCGAAAATCGTGGGC 3' (SEQ ID

NO: 43)

3' CGCCACCGTCGCCATCGCCACGCCGCGGCTTTTAGCACCCGCCGG 5' (SEQ ID NO: 44)

There are two Sac2 sites in the HBP gene between Eag1 and Avr2 so one has to make sure the vector fragment from pHW 1380 is cut at Sac2 Ubi and not at any of the other sites.

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),

2 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0; Buffer B: bufferA + 100 mM NaCl (lysis buffer); Buffer C: buffer A + 0.1% triton X-100 (wash buffer); Buffer D: buffer A + 6 M guanidinum chloride, 10 mM dithiothreitol (DTT) (extraction buffer)

Buffer E: 20 mM Tris, 6 M urea, 2 mM EDTA, 10 mM DTT, pH 7.2; Buffer F: buffer E + 0.6 M NaCl; Buffer G: 50 mM Tris-HCl, pH 8.0

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. In case of the Achromobacter lyticus protease site, 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

Example 8. Refolding of HBP by size-exclusion chromatography 0.1 to 1 mg/ml protein in urea or guanidinium chloride containing buffer (buffer D or E, see example 6) is loaded on a Sepherdex 75 HR 10/30 column (Pharmacia). The column is pre-equilibrated with refolding buffer containing a reducing/oxidising agent (e. g. 3 mM reduced glutathione and 0,3 mM oxidised glutathione in 20-100 mM Tris-sulphate, pH 7-8.5, 1-100 mM MgSO₄ and 1-30% glycerol). Both MgSO₄ and glycerol are known from crystallisation experiments to increase the solubility of HBP. 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.

Example 9. Refolding of HBP in the presence of artificial chaperones

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 sample of HBP in one of the above buffers is diluted to 10-500 μg 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

1.0 mM acetyltrimethylammonium bromide (CTAB) or 8.1 mM sodium dodecylsulphate (SDS)) are added. After incubation for 16-20 h at 4-20 °C, the stripping of the detergent is performed by mixing the sample with an equimolar amount of cyclodextrin. Purification of the protein from the reaction mixture is done as follows: first a gel filtration on Sephadex G-25, thereafter an affinity chromatography on an aprotinin column, and finally (optionally) a HIC- purification/ion-exchange chromatography. The analytical control of the final product is done as in Example 7.

Example 10. Co-solvent assisted refolding of HBP Refolding is performed as described in Example 8 with special organic modifiers included in the refolding buffer, e. g. polyethylene glycol (PEG) MW 3000. Urea or guanidinum chloride is used as denaturant. The final concentration of PEG in the refolding buffer is 2-10 times higher than the concentration of the protein. Purification of the refolded protein is carried out as described in Example 9.

Table 1

Table 1 shows the relevant contents of the plasmids used in the examples above: pHW 1280 met HBP pHW 1283 met pro Entero HBP

pHW 1288 Thio Entero met HBP pHW 1289 Thio Entero HBP pHW 1290 Thio Entero pro HBP pHW 1306 Thio Entero HBP (K6R)

pHW 1295 GST FXa HBP pHW 1296 GST THROM pro HBP

pHW 1311 PC Sma HBP pHW 1312 PC Sma HBP C98, C103, K99, K104 mut. in PC

pHW 1380 MKH8 UBI FXa HBP pHW 1383 MKH8 UBI ALP HBP (K6R)

Protease sites :

FXa : IEGR / Thrombin : LVPR / GS Enterokinase : DDDDK / ALP : A.lyticus protease : K /

Claims

ClaimsWhat is claimed is:

1. 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

d) lysing the cells of step (c);

2. A method for producing a recombinant heparin-binding protein (HBP) in bacterial cells comprising

d) lysing the cells of step (c); e) obtaining the expressed fusion protein in the insoluble fraction of the host cell lysate of step (d);

f) dissolving the obtained fusion protein of step (e) in aqueous solution;

g) cleaving the solved fusion protein of step (f);

h) purifying HBP after the cleavage of step (g), and optionally refolding HBP obtained after purification.

3. A method for producing a recombinant heparin-binding protein (HBP) in bacterial cells comprising,

b) dissolving the fusion protein in aqueous solution;

c) cleaving the solved fusion protein of step (b);

d) purifying HBP after the cleavage of step (c);

e) optionally refolding HBP obtained after purification of step (d).

4. The method according to claim 3, wherein step a) comprises

a1) obtaining transformed bacterial host cells, wherein said cells have expressed the fusion protein and accumulated said protein in the cytoplasm in insoluble form, and

a2) precipitating the fusion protein by centrifugation of the lysate of step a1 ).

5. A method for producing in bacterial cells a mammalian heparin-binding protein (HBP)

comprising, a) culturing a recombinant bacterial cell transformed by an expression vector including a hybrid gene comprising a nucleic acid sequence encoding HBP, which is is fused in frame to a sequence encoding a protease cleavage site, which in turn is fused in frame to a sequence encoding a second polypeptide in a suitable culture medium under conditions permitting expression and accumulation of said fusion protein in a form of inclusion body in the cytoplasm of said cells;

b) isolating the inclusion body of (a);

c) dissolving said inclusion body in an aqueous solution;

d) cleaving the dissolved fusion protein of (c);

f) purifying HBP after the cleavage of (d);

e) optionally refolding the purified HBP of (f).

6. The method of claim 5, wherein the inclusion body is isolated from the whole cell lysate of recombinant bacteria prepared by sonication of said cells.

7. The method of claim 5, wherein the inclusion body is collected in precipitated fraction of the lysate of claim 6 after centrifugation of said lysate at 10,000 g for 10 min.

8. The method of claim 5, wherein the inclusion body is isolated from the precipitate of claim 7 by dissolving said precipitate in aqueous solution containing a detergent.

9. The method of claim 5, wherein the inclusion body dissolved according to the claim 8 is digested by a protease selected according to the protease cleavage site expressed in the fusion protein.

10. The method of any of the claims 2, 3, or 5, wherein HBP is purified by chromatography of the digested fusion protein of claim 9, and optionally refolded thereafter.

11 The method of any of the claims 1 , 2, 3, 4 or 5, wherein HBP is a human heparin- binding protein (hHBP), which has at least about 80%, more preferably at least about 90 %, and even more preferably at least about 95 %, and most preferably at least 97% identity with the amino acid sequence set forth in SEQ ID NO:1, 3, 5, 7 and 9, natural or synthetic variants, or peptide fragments thereof.

12. The method of any of the claims 1, 2, 3, 4 or 5, wherein HBP is a porcine heparin-binding protein (pHBP), which has at least about 80%, more preferably at least about 90 %, and even more preferably at least about 95 %, and most preferably at least 97% identity with the amino acid sequence set forth in SEQ ID NO: 11 , 13, 15 and 17, natural or synthetic variants, or peptide fragments thereof.

13. The method of any of the claims 1 , 2, 3, 4 or 5, wherein HBP is encoded by a nucleic acid sequence which hybridises to the nucleic sequence set forth in SEQ ID

NO: 2, 4, 6, 8, 10, 12, 14, 16 and 18, or allelic or natural variants thereof.

14. The method of any of the claims 1 , 2, 3, 4 or 5, wherein the host cell is selected from the group comprising Bacillus subtilis, Bacillus stearothermophilus, Bacillus brevis, Bacillus lentis, Bacillus licheniformis, Streptomyces lividans, Streptomyces murinus, Pseudomonas sp, and Escherichia coli.

15. The method of any of the claims 1 , 2, 3, 4 or 5, wherein the host cell is selected from the group comprising Bacillus subtilis, and Escherichia coli.

16. The method of claim 1 , wherein the host cell is Escherichia coli.

17. The method of any of the claims 1, 2, 3 or 4, wherein HBP is expressed in a host cell of bacterium as an insoluble fusion protein.

18. A recombinant expression vector including a DNA construct comprising a DNA sequence encoding HBP, wherein said sequence is a nucleic acid sequence which hybridises to the nucleic sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 and 18, 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.

19. A DNA construct comprising a DNA sequence encoding HBP, wherein said sequence is a nucleic acid sequence which hybridises to the nucleic sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 and 18, 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.

20. The DNA construct of claim 19, wherein the DNA sequence encoding a second polypeptide is a sequence which hybridises to the DNA sequence of calf chymosin, or allelic or natural variants thereof.

21. The DNA construct of claim 19, wherein the DNA sequence encoding a second polypeptide is a sequence which hybridises to the DNA sequence of bacterial thioredoxin, or allelic or natural variants thereof.

22. The DNA construct of claim 19, wherein the DNA sequence encoding a second polypeptide is a sequence, which hybridises to the DNA sequence of ubiquitin, or allelic or natural variants thereof.

23. The DNA construct of claim 19, wherein the DNA sequence encoding a protease cleavage site is selected from the group comprising the Achromobacter lyticus, Factor Xa protease, enterokinase, or thrombin cleavage site encoding DNA sequences.

24. The DNA construct of claim 19, wherein the DNA sequence encoding a protease cleavage site is the Achromobacter lyticus cleavage site encoding DNA sequence.

25. 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.

26. The fusion protein of claim 25, wherein HBP is a human heparin-binding protein (hHBP), which has at least about 80%, more preferably at least about 90 %, and even more preferably at least about 95 %, and most preferably at least 97% identity with the amino acid sequence set forth in SEQ ID NO: 1 , 3, 5, 7 and 9, natural or synthetic variants, or peptide fragments thereof.

27. The fusion protein of claim 26, wherein HBP is a porcine heparin-binding protein (pHBP), which has at least about 80%, more preferably at least about 90 %, and even more preferably at least about 95 %, and most preferably at least 97% identity with the amino acid sequence set forth in SEQ ID NO: 11 , 13, 15 and 17, natural or synthetic variants, or peptide fragments thereof.

28. The fusion protein of claim 25, wherein the second polypeptide sequence is a heterologous polypeptide sequence.

29. The fusion protein of any of the claims 25, 26, 27 or 28, wherein HBP is positioned C-terminally in the fusion protein and the second polypeptide sequence is positioned N-terminally in the fusion protein.

30. The fusion protein of claim 30, wherein the second polypeptide is a calf chymosin.

31. The fusion protein of claim 30, wherein the second polypeptide is a bacterial thioredoxin.

32. The fusion protein of claim 30, wherein the second polypeptide is ubiquitin.

33. The fusion protein of claim 25, wherein the protease cleavage site is an amino acid sequence which can be cleaved by a protease selected from the group comprising the Achromobacter lyticus and Factor Xa proteases, enterokinase, or thrombin.

34. The fusion protein of claim 25, wherein the protease cleavage site is an amino acid sequence of the Achromobacter lyticus protease cleavage site.

35. Use of the fusion protein as defined in any of claims 25-38 for producing a heparin-binding protein (HBP).

36. Use of a heparin-binding protein (HBP) produced as defined in any of the claims 2-17 for the preparation of a medicament.