WO1997033984A1

WO1997033984A1 - Novel achromobacter lyticus protease variants

Info

Publication number: WO1997033984A1
Application number: PCT/DK1997/000100
Authority: WO
Inventors: Asser Andersen; Per Balschmidt; Sven Branner; Sven Hastrup
Original assignee: Novo Nordisk A/S
Priority date: 1996-03-12
Filing date: 1997-03-07
Publication date: 1997-09-18
Also published as: ZA972083B; AU2091397A

Abstract

The invention relates to novel Achromobacter lyticus protease I variants wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 have been replaced by another amino acid residue which can be encoded by nucleic acid constructs, or wherein one or more of the lysine residues in the above-mentioned positions or other amino acid residues introduced into the above-mentioned positions have been chemically modified. The invention furthermore relates to nucleic acid constructs encoding said protease variants, and vectors and host cells comprising said constructs. The protease variants are useful for cleaving Lys-X peptide bonds in polypeptides, such as preproinsulins.

Description

NOVEL ACHROMOBACTER LYTICUS PROTEASE VARIANTS

FIELD OF INVENTION

The present invention relates to novel polypeptides with Achromobacter lyticus protease (API) activity, nucleic acid constructs encoding the polypeptides, and recombinant vectors and recombinant host cells comprising the nucleic acid constructs.

BACKGROUND OF THE INVENTION

The earliest studies of proteolytic enzymes (proteases) centered around the important digestive enzymes Pepsin, Trypsin and Chymotrypsin. Today, many proteases of animal, plant and microbial origin are known and several have found sufficient application to be produced industrially. These applications include applications for medical purposes, for food processing and laundering purposes as well as applications in protein synthesis and structural analysis.

Proteolysis catalyzed by proteases is characterized by specificity, high yield and stability of the product (peptide fragments and amino acids) to the reagent (protease).

Lysyl Endopeptidase is a protease secreted by the soil bacterium Achromobacter lyticus (cf. Masaki et al., Agric.Biol.Chem. 42, 1978, p. 1443). The protease is identical to the enzyme discovered by Masaki et al., and named Achromobacter protease I (cf. Masaki et al., Biochem.Biophvs.Acta 660, 1981, p. 44). Japanese patent application No.64-02574 (Wako) (priority from 870622) relates to a method for purification of Achromobacter protease I. The primary structure of Achromobacter lyticus protease I appears from S. Tsunasawa et al., J.Biol.Chem. 264(7), 1989, pp. 3832-3839. API is a trypsin-like serine protease which specifically cleaves the peptide bonds (-Lys-X-) at the side of the carboxyl groups of lysine residues in proteins and peptides, and is also called a lysyl endopeptidase (EC 3.4.21.50). This enzyme cleaves all Lys-X bonds including the Lys-Pro bond, and therefore has been used for the fragmentation of proteins or peptides for their primary structural analysis, the preparation of peptide maps, and the synthesis of Lys-X-compounds. Furthermore, API is presently used for cleaving fusion proteins or producing des-B30-insulin. European patent No. 17.938 (Shionogi) (priority from 790413) relates to a method for producing B30-Thr-insulin by reacting a derivative of threonine and des-B30-insulin with a protease, e.g. A.lyticus protease I. Japanese patent application No. 57-67548 (Shionogi) (priority from 801014) relates to a method for producing insulin analogues in which the B30 amino acid residue has been substituted with another amino acid residue by using trypsin, a trypsin-like protease or A.lyticus protease I. European patent No. 92.829 (Wako) (priority from 820423) relates to the preparation of human insulin derivatives from porcine insulin by using A.lyticus protease I or a water-soluble cross-linked A.lyticus protease I. European patent application No. 367.302 (Wako) (priority from 820423) relates to a new water soluble, cross- linked form of Achromobacter protease I useful for converting pig insulin to human insulin by specific removal and replacement of the B30 amino acid. The protease is cross linked with a water-soluble protein. Japanese patent No. 92-076673 (Wako) (priority from 831202) relates to a novel enzyme produced by cross-link polymerizing the Achromobacter protease I with polyalkyleneglycol. European patent applications Nos. 206.769 and 440.311 (Fujisawa) (priority from 850620) relates to the use of Achromobacter protease I for cleaving fusion proteins having a lysine between a protective peptide and a target peptide (e.g. human atrial natriuretic polypeptide (hANP)) containing no lysine residues. European patent No. 354.507 (Hoechst) (priority from 880810) relates to a method for producing des-B30-insulin by using a protease, e.g. A.lyticus protease I.

Since natural API is isolated in only very small amounts from Achromobacter lyticus, gene engineering techniques has been applied and a method for producing API in larger amounts has been developed. The gene encoding A.lyticus protease I has been cloned in E. coli and API produced by culturing transformed microorganisms (cf. T. Ohara et al., J.Biol.Chem. 264(34). 1989, pp. 20625-20631).

European patent application No. 387.646 (Wako) (priority from 890314) relates to a novel DNA encoding Achromobacter protease I for recombinant production of enzyme, and for fragmentation of protein(s) and peptide, for peptide mapping and synthesis of Lys-X- compounds. WO 96/17943 (Novo Nordisk A/S) (priority from 941209) relates to a method of producing an extracellular protein, e.g. API, in a bacterium transformed with a DNA sequence encoding the API. One object of this invention is to make available an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a higher activity than Achromobacter lyticus protease I.

A further object of this invention is to make available an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a higher stability than Achromobacter lyticus protease I.

A further object of this invention is to make available an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a better performance than Achromobacter lyticus protease I when immobilized on a water-insoluble earner.

SUMMARY OF THE INVENTION

It has now been found that a modification of the lysine residues in Achromobacter lyticus protease I (API, cf. Fig. 1) leads to an increased efficiency in cleaving Lys-X peptide bonds in peptides and proteins compared to the "native" API.

Furthermore, it has now been found that the modification of the lysine residues in API leads to increased stability of the enzyme by diminishing the autoproteolysis.

Furthermore, it has now been found that the modification of the lysine residues in API leads to increased performance rates when immobilized on a water-insoluble carrier.

Furthermore, it has now been found that the modification of the lysine residues in API leads to an increased specific activity of the protease variant.

In the present context the term "Achromobacter lyticus protease I variant" designates an enzyme which specifically splits a protein or peptide at the C-terminal end of lysine and which has a homology with Achromobacter lyticus protease I of more than 90%, preferably more than 95%. Accordingly, the present invention relates to novel Achromobacter lyticus protease I variants wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 (referring to Fig. No.1) have been replaced by another amino acid residue which can be encoded by nucleic acid constructs, or wherein one or more of the lysine residues in the above mentioned positions or other amino acid residues introduced into the above mentioned positions have been chemically modified.

In another aspect, the present invention relates to a A.lyticus protease I variant immobilized on a water-Insoluble carrier with or without the use of a spacer or linker molecule.

In another aspect, the present invention relates to a cross-linked polymer of a A.lyticus protease I variant, or a protease variant cross-linked to a carrier protein with or without the use of a spacer or linker molecule.

In another aspect, the present invention relates to nucleic acid constructs comprising a nucleotide sequence, preferably a DNA sequence, encoding said Achromobacter lyticus protease variants.

In a further aspect, the present invention relates to recombinant vectors comprising the said nucleic acid constructs.

In a still further aspect, the present invention relates to recombinant host cells comprising said nucleic acid constructs or said vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig.1 shows the amino acid sequence of unmodified API.

Fig.2 shows the gene structure of the unmodified A.lyticus protease I gene.

Fig.3 A-C show the general amino acid sequence of the novel API-derivates according to the invention, where Xaa designates an amino acid residue which may be encoded by a nucleic acid construct, or Xaa designates a lysine residue which has been chemically modified, and, in particular, [K30RJ-API (SEQ ID NO:1) and [Me_rK30]-API (SEQ ID NO:2).

Fig.4 A-B show plasmid charts of A.lyticus protease I expression plasmid, pSX547, and A.lyticus protease I K30R expression plasmid, pSX582.

Fig.5 shows the processing of preproinsulin into des-B30-insulin using methylated API ([Me K30J-API).

Fig.6 shows the processing of preproinsulin into des-B30-insulin using [K30RJ-API.

Fig.7 A-B shows plasmid charts illustrating the preparation of A.lyticus protease I expression plasmid pSX547.

DETAILED DESCRIPTION OF THE INVENTION

In the Achromobacter lyticus genome, the gene coding for A.lyticus protease I encodes a polypeptide of 653 amino acid residues. This includes a signal (or pre) peptide of 20 amino acids, an N-terminal propeptide of 185 amino acids, a core (or mature) protein of 268 amino acids which is the active protease, and a C-terminal propeptide of 180 amino acids, as shown in figs. 1 and 2 (cf. T.Ohara et al., J.Biol.Chem. 264 (34), 1989, pp. 20625-20631).

It has now been found that if the active protease (API, cf. Fig. 1) is modified at the lysine residues, the specific activity of the protease is increased by a factor of about 2 to 3. Furthermore, the modification of lysine residues in API leads to increased stability of the enzyme by diminishing the autoproteolysis.

Accordingly, the present invention relates to novel Achromobacter lyticus protease I variants wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 (referring to Fig. 1) have been replaced by another amino acid residue which can be encoded by nucleic acid constructs, or wherein one or more of the lysine residues in the above mentioned positions or other amino acid residues introduced into the above mentioned positions have been chemically modified. In another embodiment the present invention relates to Achromobacter lyticus protease I denvatives compnsing the ammo acid sequence shown i Fig 1 wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 are - replaced by an ammo acid residue different from lysine which is positively charged at neutral pH,

- or replaced by an ammo acid residue which is negatively charged at neutral pH,

- or replaced by a hydrophilic amino acid residue which is uncharged at neutral pH,

- or replaced by a hydrophobic ammo acid residue which is uncharged at neutral pH, - or replaced by a modified lysine residue with the general formula

[-NH-CH[(CH₂)₄-NRR ]-CO-], in which

R is hydrogen or-C(1-6)-alkyl, and R¹ is -C(1-6)-alkyl, or-CNH-NR'R", or-CNH-R', where R' and R" are different or identical, and are H or C(1-6)-alkyl,

where R² is C(1-6)-alkyl, or -CH_rCH_rCOOH, or -CH=CH-COOH

When the lysine residues are chemically modified the N-terminal ammo group may be modified as well

In a preferred embodiment of the invention, one or more of the lysine residues in the API protease are replaced by an ammo acid which is negatively charged at neutral pH, e g Asp, or Glu, or replaced by a modified lysin residue with the general formula [-NH-CH[(CH₂)₄-NRR¹]-CO-j, in which R is hydrogen or C(1-6)-alkyl, and R¹ is -C(O)-R², where R² is -CHrCHrCOOH, or -CH=CH-COOH

The term "C(1-6)-alkyl" as used herein, alone or in combination, refers to a straight or branched, saturated hydrocarbon chain having 1 to 6 carbon atoms, such as e g methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, 3- methylbutyl, n-hexyl, 4-methylpentyl, neopentyl, and 2,2-dιmethylpropyl

In a further preferred embodiment one or more of the lysine residues are replaced by a hydrophilic ammo acid which is non-charged at neutral pH, e g Gly, Asn, Gin, Ser, or Thr, or replaced by a lysine residue with the formulae [-NH-CH[(CH₂)₄-NRR¹]-CO-], in which R is hydrogen or C(1-6)-alkyl, and R¹ is -CO-NR'R", where R' and R" are different or identical, and are hydrogen or C(1-6)-alkyl, or R¹ is -CO-R² , where R² is C(1-6)-alkyl.

In another preferred embodiment one or more of the lysine residues in the API protease are replaced by a hydrophobic amino acid which is uncharged at neutral pH, e.g. He, Leu, Val, Ala, Phe, Tyr, Tip, Pro, Cys, or Met.

In another more preferred embodiment one or more of the lysine residues in the API protease are replaced by an amino acid different from lysine which is positively charged at neutral pH, e.g. Arg, or His, or replaced by a modified lysine residue with the general formula [-NH- CH[(CH₂)₄-NRR¹]-CO-], in which R is hydrogen or C(1-6)-alkyl, and R is C(1-6)-alkyl, or -CNH- NR'R", or -CNH-R', where R' and R" are different or identical, and are hydrogen or C(1-6)- alkyl.

In the most preferred embodiment one or more of the lysine residues in the API protease are replaced by an arginine residue or a residue with the general formula [-NH-[(CH₂)₄-NMe₂]-CO-].

In a further preferred embodiment the lysine residue in position 30 is modified.

In a further preferred embodiment the lysine residue in position 49 is modified.

In a further preferred embodiment the lysine residue in position 106 is modified.

In a further preferred embodiment the lysine residue in position 155 is modified.

In a further preferred embodiment the lysine residue in position 203 is modified.

In the most preferred embodiment the protease is [K30RJ-API (SEQ ID NO:1) or [Me₂-K30]-API (SEQ ID NO:2).

The amino acid in position 30 in API is probably the most readily accessible lysine residue from an sterical and electrostatically point of view. The distance from this lysine residue to the active site of the enzyme is rather short (approximately 0.9 nm). This indicates that this amino acid in particular is very important for the accessibility of substrate to the active site of the enzyme, and that the amino acid in position 30 in particular has influence on the specific activity of the enzyme.

The Achromobacter lyticus protease variants according to the present invention are useful for cleavage of polypeptides. Polypeptides suitable for cleavage may be fused polypeptides, or polypeptides such as preproinsulins or analogues hereof, preferably preproinsulins. The preproinsulins may be of human, porcine, or bovine origin, preferably human preproinsulin.

The modified protease may be used in solution; alternatively, the protease may be cross- linked with conventional cross-linking agents (for instance glutaraldehyde, diisocyanate, formaldehyde, etc.) to convert it into a polymer; or the protease may be cross-linked to a carrier polypeptide. The protease may be immobilised on a soluble matrix, such as a polyglutamic acid matrix, by conventional procedures being well known to persons skilled in the art.

Alternatively, the modified API may be immobilized on a solid phase when used for cleavage of polypeptides. The solid phase may be a cellulose matrix, a polysaccharide matrix such as a cellulose or cross-linked agarose matrix, a silica matrix, a dextran matrix, a polyacrylamide matrix, a polyamide matrix, (e.g. sephadex or sepharose), preferably a polysaccharide matrix. Examples of preactivated polysaccharide matrixes include Mini-Leak™ and CNBr- Sepharose™ Examples of preactivated polyacrylamide matrixes include Eupergit™ and Affi-

Gel TM

Conventional procedures for immobilizing an enzyme on a water-insoluble carrier may be employed, such methods being well known to persons skilled in the art. When immobilized or cross-linked, the enzyme may be attached to the matrix or carrier protein via a spacer or linker. Such a spacer or linker may be a polyfunctional organic compound. Suitable spacers or linkers are well known to persons skilled in the art, as are the methods for employing such spacers or linkers. The protease according to the present invention may be produced by post-translational chemical modification of the "natural" protease, or by site directed mutagenesis of the A.lyticus protease encoding nucleic construct.

Chemical modifications of lysine residues

Lysine is an amino acid having a second amino entity located in ε-position. Such amino groups are basic groups and are positively charged except at high pH. In uncharged form amino groups are powerful nucleophiles and are capable of being chemically modified in several ways, such well known modifications being, e. g., reaction with carboxylic acid anhydride (e.g. acetic acid anhydride, succinic acid anhydride, maleic acid anhydride), reaction with cyanate (carbamylation) giving substituted ureas, reaction with isoureas (guadination), reaction with imidates (aminidation), and reductive alkylation.

Chemical modifications of peptides and proteins are well known to the persons skilled in the art (cf. Chemical Modification of Proteins, G.E.Means and R.E.Feaney, Holden-Day, Inc., 1971).

Site directed mutagenesis

The procedures used for preparing a DNA construct with specific, site directed changes (Site Directed Mutagenesis) are well known to persons skilled in the art (cf. "Splicing by extension overlap", Horton et al.. Gene 77. 1989, pp. 61-68).

Likewise are procedures for preparing a DNA construct using polymerase chain reaction using specific primers well known to persons skilled in the art (cf. PCR Protocols, 1990, Academic Press, San Diego, California, USA).

Nucleic acid construct

As used herein the term "nucleic acid construct" is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The term "construct" is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest. The construct may optionally contain other nucleic acid segments. The nucleic acid construct of the invention encoding the polypeptides of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

The nucleic acid construct of the invention encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805. According to the phospho¬ amidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.

Furthermore, the nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.

The nucleic acid construct may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described by Sambrook et al., supra, or as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491. For instance, it may be envisaged that the DNA sequence encoding the prepropeptide may be prepared by PCR amplification of chromosomal DNA of the species from which the the prepropeptide is derived. Likewise, the DNA sequence encoding the desired protein may be prepared by PCR amplification of chromosomal DNA of the species from which the protein is derived, or for instance by screening a genomic or cDNA library with oligonucleotides as indicated above.

In another preferred embodiment, the nucleic acid construct of the invention comprises nucleic acid sequences encoding the amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2. The nucleic acid construct is preferably a DNA construct which term will be used exclusively in the following.

Recombinant vector In a further aspect, the present invention relates to a recombinant vector comprising a DNA construct of the invention. The recombinant vector into which the DNA construct of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and 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 extrachromosomal entity, the 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.

The vector is preferably an expression vector in which the DNA sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide.

In a preferred embodiment of the invention, the expression vector is the plasmid pSX582 as described in Example 2 and Fig.4B.

The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the polypeptide of the invention in mammalian cells are the SV40 promoter (Subramani et al., MoL Cell BjoL 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter. Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J BjoL Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J, MoL Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPM (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters. Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus subtilis xylosidase gene, or by the phage Lambda P_R or P_L promoters or the E. coli lac, tijj or tac promoters.

The DNA sequence encoding the polypeptide of the invention may also, if necessary, be operably connected to a suitable terminator. The vector may further comprise elements such as polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences

The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a polypeptide of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the polypeptide in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide. The secretory signal sequence may be that normally associated with the polypeptide or may be from a gene encoding another secreted protein. For secretion from yeast cells, the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed polypeptide into the secretory pathway of the cell. The signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the α-factor signal peptide, the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L.A. Vails et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

For efficient secretion in yeast, a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the polypeptide. The function of the leader peptide is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The leader peptide may be the yeast α-factor leader (the use of which is described in e.g. US 4,546,082, EP 16 201, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463, WO 92/11378, or WO 95/34666.

The procedures used to ligate the DNA sequences coding for the present polypeptide, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.).

Host cells

The DNA sequence encoding the present polypeptide introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another seαetory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a cDNA sequence encoding a polypeptide native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present polypeptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of bacterial host cells which, on cultivation, are capable of producing the polypeptide of the invention are grampositive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearother ophilus, B. al alophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gramnegative bacteria such as Echerichia coli. The transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra). In a preferred embodiment, the transformed host cell is Echerichia coli.

When expressing the polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide.

Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. MoL BjoL 159 (1982), 601 - 621 ; Southern and Berg, JL MoL Appl. Genet. 1 (1982), 327 - 341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422 - 426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO 1 1 (1982), 841 - 845.

Examples of suitable yeasts cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are described, e.g. in US 4,599,311, US 4,931,373, US 4,870,008, 5,037,743, and US 4,845,075, all of which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine. A preferred vector for use in yeast is the POT1 vector disclosed in US 4,931,373. The DNA sequence encoding the polypeptide of the invention may be preceded by a signal sequence and optionally a leader sequence , e.g. as described above. Further examples of suitable yeast cells are strains of Saccharomyces sp., including Saccharomyces cerevisiae, Saccharomyces kluyveri, and Saccharomyces uvarum; Schizosaccharomyces pombe; Kluyveromyces sp., including Kluyveromyces lactis; Hansenula sp., including Hansenula polymorpha; Pichia sp., including Pichia pastoris, Pichia methanolica, and Pichia kluyveri; Yarrowia lipolytica; Candida sp., including Candida utilis, and Candida cacaoi; Geotrichum sp.; and Geotrichum fermentans (cf. Gleeson et al., J, Gen. Microbiol. 132, 1986, pp. 3459- 3465; US 4,882,279).

Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.

When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination.

The transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the present polypeptide, after which the resulting polypeptide is recovered from the culture.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suit¬ able media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The polypeptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.

The invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed.

EXAMPLES

Example 1

Reductive methylation of API

Essentially the reduction was carried out according to Means and Feaney, Chemical Modification of Proteins, Holden-Day, Inc. (1971), p.217.

6 ml of a 31 mg/ml solution of purified API was diluted with 50 ml 0.2 M borate buffer, having a pH value of 9, and cooled on ice. (200 ml reference sample was taken). 27.5 mg NaBH₄ was added to the cold enzyme solution and 5 portions of each 25 ml 37% formalin was added at 6 minutes intervals. The total amount of formalin was added during approximately 40 minutes. (200 ml reaction No.1 sample was taken.)

The reaction was repeated with additionally 25 mg NaBH₄ and 5 times 25 ml 37% formalin during a 55 minutes period. (200 ml reaction No.2 sample was taken). To quench the reaction, the main part of the reaction mixture was adjusted to pH 6.3 with 1 M acetic acid.

The activity in the samples taken was determined by using the amidolytic assay described in Example 3. The reactivities of the sample taken prior to reaction, and the sample taken after reactions Nos.1 and 2 was compared: Sample Units/ml Relative reactivity

Reference 15.8 1.00

Reaction No.1 25.4 1.61

Reaction No.2 29.1 1.84

The relative reactivity was measured against the substrate z-Lys-pNA, cf. Example 3.

Example 2 Site-directed-mutagenesis of API:

The lysine in position 30 of the mature enzyme was substituted with an arginine using the method "Splicing by overlap extension", Horton et al., Gene 77, 1989, pp. 61-68. Sequence positions in the following is taken from Ohara et al., J.Biol.Chem. 264 (34), 1989, pp. 20625- 20631.

Two PCR products were made. One going from position 827 to 1100 (primer MHJ 3989 and 4003). The other going from position 1080 to 1361 (primer MHJ 4011 and 3990). Primers MHJ 3989 and 3990 overlap with the Asc I site in the position 836 and the Xho I site in position 1352, respectively. Primers 4003 and 4011 are complementary and overlap with the codon for amino acid 30, changing this from AAG (lysine) to CGA (arginine). This introduces a Nru I site into the sequence which can be used to distinguish between the mutant and the wild type.

The two PCR products were mixed and denatured. Taq polymerase was added. The product was reannealed at 50°C and extended for 4 minutes at 72°C. The primers MHJ 3989 and 3990 were added and the DNA was amplified for 20 cycles. The resulting PCR product was cut with Asc I and Xho I and cloned into pSX547, where the wild type Asc I - Xho I fragment had been removed. The resulting plasmid pSX582, was transformed into E.coli 3110 lac l^q; the resulting strain was plated onto LB-plates with 200 mg/ml ampicillin (J.H. Miller (1972), Experiments in Molecular genetics, Cold Spring Harbor Laboratory).

The resulting strain was grown in liquid LB medium containing 0.4% lactose for 44 hours at 26mC whereafter the culture was centrifuged and the supernatant tested for lysyl- endopeptidase activity with Benzoyl-lysyl-pNA. The result was positive. The mutant enzyme was recovered from the supernatant.

Primers: MHJ 3989: 5'-CAA CTC GGC GCG CCA ACT GTG GAC-3'

MHJ 3990: 5^*-TGT TCA ACT CGA GCA GGG TGA AGT C-3'

MHJ4003: 5'-CGTGCCGCTTCGCGAGTACGCACCGACCGCGC-3'

MHJ4011: 5'-GCGTACTCGCGAAGCGGCACGCTGGCCTGTACC-3'

Bold letters: Mutated codon. Underlined: Nru I restriction site.

Example 3

Site-directed mutagenesis of API: API with lysines at position 30 and 49 mutated to arginine:

The mutant described in example 1 was used as template, resulting in a double mutant with both lysines in position 30 and 49 substituted with arginines. The lysine in position 49 of the mature enzyme was substituted with an arginine using Stratagenes "QuickChange™ Site-Directed Mutagenesis Kit". Sequence positions in the following is taken from Ohara et al.. J.Biol.Chem. 264 (34), 1989, pp. 20625-20631.

Two complementary primers going from position 1006-1037 were made (primer EliA 9 and 10). These primers overlap with the codon for amino acid 49, changing this from AAG (lysine) to CGT (arginine). Primers EliA9 and 10 were mixed with the template plasmid pSX582. Pfu polymerase was added and and the mixture denatured. Subsequently 18 cycles were performed as follows: denature 45 seconds at 97°C, anneal 1 minute at 55°C and extend 8 min at 68°C. Finally the mixture was digested with Dpn1 for 1 hour at 37°C to remove template (pSX582). The resulting double stranded nicked plasmids were transformed into E.coli and plated onto LB-plates with 200 μg/ ml ampicillin. Colonies were picked and a plasmid containing the desired mutation was identified by DNA sequencing. This was named pEA186. The resulting strain was grown at 26°C in liquid LB medium to an OD600 of 1.5. Subsequently 0.4% lactose was added and the growth continued for 44 hours whereafter the culture was centrifuged and the supernatant tested for lysyl-endopeptidase activity with Benzoyl- lysyl-pNA. The result was positive. The mutant enzyme was recovered from the supernatant.

Primers

EliA 9: 5'-CCG CCA ACG ACC GCC GTA TGT ACT TCC TGA CC-3"

EliA 10: 5'-GGT CAG GAA GTA CAT ACG GCG GTC GTT GGC GG-5"

Bold letters: mutated codon

Example 4

Site directed mutagenesis of API: API with lysines at position 30 and 106 mutated to arginine:

The mutant described in example 1 was used as template, resulting in a double mutant with both lysines in position 30 and 106 substituted with arginines. The lysine in position 106 of the mature enzyme was substituted with an arginine using Stratagenes "QuickChange™ Site-Directed Mutagenesis Kit". The mutation was introduced using the method described in Example 3, except that the complementary primers are going from position 851 to 882 (primer EliA 11 and 12). These primers overlap with the codon for amino acid 106, changing this from AAG (lysine) to CGT (arginine). The resulting plasmid was named pEA187. The supernatant from the resulting culture was tested for API activity and found to be positive as described in example

Primers

EliA 11 : 5 -CGG GTT CGA CGG TCC GTG CGA CCT ACG CCA CC-3'

EliA 12: 5'-GGT GGC GTA GGT CGC ACG GAC CGT CGA ACC CG-3'

Bold letters: mutated codon

Example 5 Site directed mutagenesis of API: API with lysines at position 30, 46 and 106 mutated to arginine:

Plasmid pEA187 was cut with restriction enzymes Pstl and BspMI. This results in a DNA fragment of 0.4 kb containing the code for arginine at position 106 of API. Plasmid pEA186 was cut with the same restriction enzymes. A DNA fragment of 3.8 kb was isolated and ligated with the 0,4 kb fragment, resulting in a plasmid pEA189 encoding an API where lysines at position 30, 49 and 106 has been mutated to arginine. The resulting plasmid was named pEA189.

The supernatant from the resulting culture was tested for API activity and found to be positive as described in Example 6.

Example 6 Amidolytic assay for Achromobacter lyticus lysyl specific endopeptidase (API):

Materials and solvents:

N°-(Benzyloxycarbonyl)-L-lysine-p-nitroanilide (Z-Lys-pNA) hydrochloride 0.1 M tris(Hydroxymethyl)aminomethane/HCI buffer, pH 8.0 Dimethyl sulfoxide (DMSO)

ELISA plate (96 wells) ELISA reader (e.g. Bio-Tek™ EL 340)

Solutions:

Substrate solution:

A stock substrate solution containing 0.004 M Z-Lys-pNA in 0.1 M tris/HCI buffer, pH 8.0, is prepared by dissolution of 100 mg Z-Lys-pNA,HCI in 1 ml of DMSO and subsequent dilution to 57 ml with tris/HCI buffer. The pH value in the resulting solution is readjusted to 8.0.

Standard enzyme solution: 1.0 ml of water is added to a vial containing 10 units of lyophilized API (WAKO Pure

Chemicals Ltd., Japan). Before test, a sample of the solution is diluted x 400 with tris/HCI buffer.

Test enzyme solution: The enzyme solution to be tested is suitably diluted with tris/HCI buffer, pH 8.0.

Procedure:

100 ml test solution and 100 ml substrate solution are mixed in an ELISA well and the optical density (OD) at 405 nm is measured each minute during 10 minutes at room temperature (21 °C) in an ELISA reader. The DOD^_m/min is calculated and the activity relative to the standard is calculated using the DOD+oβ_nm min from a corresponding test with the standard enzyme solution. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novo Nordisk A/S

(B) STREET: Novo Alle

(C) CITY: 2880 Bagsvaerd (E) COU NTR Y: Denmark

(G) TELEPHONE: +4544448888 (H) TELEFAX: +4544490555

(ii) TITLE OF INVENTION: Novel Achromobacter Lyticus Protease Variants (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(v) CURRENT APPLICATION DATA: APPLICATION NUMBER:

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 268 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Gly Val Ser Gly Ser Cys Asn He Asp Val Val Cys Pro Glu Gly Asp 1 5 10 15 Gly Arg Arg Asp lie He Arg Ala Val Gly Ala Tyr Ser Arg Ser Gly 20 25 30

Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg 35 40 45

Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser 50 55 60

Thr Ala Ala Ser He Val Val Tyr Trp Asn Tyr Gin Asn Ser Thr Cys 65 70 75 80

Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met 85 90 95

Ser Gin Thr Gin Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser 100 105 110 Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn

115 120 125

Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gin Asn Tyr Pro Gly Ala 130 135 140

He Ala He His His Pro Asn Val Ala Glu Lys Arg lie Ser Asn Ser 145 150 155 160

Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr 165 170 175

His Leu Asn Val Gin Trp Gin Pro Ser Gly Gly Val Thr Glu Pro Gly 180 185 190 Ser Ser Gly Ser Pro He Tyr Ser Pro Glu Lys Arg Val Leu Gly Gin

195 200 205

Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser 210 215 220

Asp Gin Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala 225 230 235 240

Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gin 245 250 255

Phe He Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro 260 265 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 268 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Gly Val Ser Gly Ser Cys Asn lie Asp Val Val Cys Pro Glu Gly Asp 1 5 10 15 Gly Arg Arg Asp He He Arg Ala Val Gly Ala Tyr Ser Xaa Ser Gly

20 25 30

Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg 35 40 45

Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser 50 55 60

Thr Ala Ala Ser He Val Val Tyr Trp Asn Tyr Gin Asn Ser Thr Cys 65 70 75 80

Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met 85 90 95 Ser Gin Thr Gin Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser

100 105 110

Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn 115 120 125

Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gin Asn Tyr Pro Gly Ala 130 135 140

He Ala He His His Pro Asn Val Ala Glu Lys Arg He Ser Asn Ser 145 150 155 160 Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr 165 170 175

His Leu Asn Val Gin Trp Gin Pro Ser Gly Gly Val Thr Glu Pro Gly 180 185 190

Ser Ser Gly Ser Pro He Tyr Ser Pro Glu Lys Arg Val Leu Gly Gin 195 200 205

Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser 210 215 220

Asp Gin Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala 225 230 235 240

Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gin 245 250 255 Phe He Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro

260 265

where Xaa in pos.30 represents the amino acid 6-N-dimethyllysine.

Claims

1. An Achromobacter lyticus protease I variant wherein one or more of the lysine residues in positions 30, 49, 106, 155 and 203 (referring to Fig. 1) have been replaced by another amino acid residue which can be encoded by nucleic acid constructs, or wherein one or more of the lysine residues in the above mentioned positions or other amino acid residues introduced into the above mentioned positions have been chemically modified.

2. An Achromobacter lyticus protease variant according to claim 1 comprising the amino acid sequence shown in Fig. 1 wherein one or more of the lysine residues in positions 30, 49, 106,

155, and 203 are

■ replaced by an amino acid different from lysine and which is positively charged at neutral pH,

■ or replaced by an amino acid which is negatively charged at neutral pH,

■ or replaced by a hydrophilic amino acid which is uncharged at neutral pH, ■ or replaced by a hydrophobic amino acid which is uncharged at neutral pH,

■ or replaced by a modified lysine residue with the general formula

[-NH-CH[(CH₂)₄-NRR¹]-CO-j, in which

R is hydrogen or C(1-6)-alkyl, and R¹ is C(1-6)-alkyl, or -CNH-NR'R", or -CO-NR'R", or -CNH-R', where R' and R" are different or identical, and are hydrogen or C(1-6)-alkyl, or R¹ is -CO-R² , where R² is C(1-6)-alkyl, or-CH₂-CH_rCOOH, or -CH=CH-COOH.

3. A protease according to any of the preceding claims 1-2 wherein one or more of the lysine residues are replaced by an amino acid which is negatively charged at neutral pH, e.g. Asp, or Glu, or replaced by a modified lysine residue with the general formula [-NH-CH[(CH₂)₄-NRR¹]-CO-], in which R is hydrogen or C(1-6)-alkyl, and R¹ is -CO-R², where R² is -CH_rCH₂-COOH, or -CH=CH-COOH.

4. A protease according to any of the preceding claims 1-2 wherein one or more of the lysine residues are replaced by a hydrophilic amino acid which is uncharged at neutral pH, e.g. Gly, Asn, Gin, Ser, or Thr, or replaced by a lysine residue with the general formula [-NH-CH[(CH₂)₄- NRR¹]-CO-], in which R is hydrogen or C(1-6)-alkyl, and R¹ is -CO-NR'R", where R' and R" are different or identical, and are hydrogen or C(1-6)-aikyl, or R¹ is -CO-R², where R² is C(1-6)-alkyl.

5. A protease according to any of the preceding claims 1-2 wherein one or more of the lysine residues are replaced by a hydrophobic amino acid which is uncharged at neutral pH, e.g. He,

Leu, Val, Ala, Phe, Tyr, Trp, Pro, Cys, or Met.

6. A protease according to any of the preceding claims 1-2 wherein one or more of the lysine residues are replaced by an amino acid different from lysine which is positively charged at neutral pH, e.g. Arg, or His, or replaced by a modified lysine residue with the general formula [- NH-CH[(CH₂)₄-NRR¹]-CO-], in which R is hydrogen or C(1-6)-alkyl, and R¹ is C(1-6)-alkyl, or - CNH-NR'R", or -CNH-R', where R' and R" are different or identical, and are hydrogen or C(1-6)- alkyl.

7. A protease according to any of the preceding claims 1-2 and 6 wherein one or more of the lysine residues are replaced by an arginine residue or a residue with the general formula [-NH- [(CH₂)₄-NMe₂]-CO-J wherein Me represents methyl.

8. A protease according to any of the preceding claims wherein the lysine residue in position 30 is replaced.

9. A protease according to any of the preceding claims which is [K30R]-API (SEQ ID NO: 1) or [Me_rK30]-API (SEQ ID NO: 2).

10. A protease according to any of the preceding claims wherein the lysine residue in position 49 is replaced.

11. A protease according to any of the preceding claims wherein the lysine residue in position 106 is replaced.

12. A protease according to any of the preceding claims wherein the lysine residue in position 155 is replaced.

13. A protease according to any of the preceding claims wherein the lysine residue in position 203 is replaced.

14. An Achromobacter lyticus protease I variant as described in any of claims 1-13 immobilized on a water-insoluble carrier with or without the use of a spacer or linker molecule.

15. The immobilized variant according to claim 14, wherein said water-insoluble carrier is a polysaccharide earner.

16. A cross-linked polymer of an Achromobacter lyticus protease I variant as described in any of claims 1-13.

17. The polymer as claimed in claim 16, wherein said enzyme is cross-linked with a cross-linking agent with or without the use of a spacer.

18. The polymer as claimed in claim 17, wherein said cross-linking agent is a polyfuπctional organic compound.

19. A nucleic acid construct comprising a nucleotide sequence encoding a polypeptide defined in any of claims 1-13.

20. A nucleic acid construct according to claim 19 comprising a nucleotide sequence encoding a polypeptide defined in claim 9.

21. A recombinant vector comprising the nucleic acid construct defined in any of claims 19-20.

22. A recombinant vector according to claim 21 where the vector is the plasmid pSX582 as described in example 2 and Fig. 4b.

23. A recombinant host cell comprising the nucleic acid construct according to any of claims 19- 20 or the vector according to any of claims 21-22.

24. A recombinant host cell according to claim 23, which is of bacterial origin.

25. Any novel feature or combination of features described herein.