Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
  • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
  • C12N9/641—Cysteine endopeptidases (3.4.22)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

• TITLE METHOD FOR OBTAINING MATURE PROTEASE BY ENZYMATIC DIGESTION
• the present invention relates to a method for obtaining a cysteine protease in mature form.
• a propolypeptide is generally inactive and can be converted to ma- ture active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide resulting in the mature active polypeptide.
• mites produce several classes or groups of allergens, one of which is known as Group 1 allergens.
• Group 1 allergens displaying considerable cross-reactivity, have been found in Dermatophagoides pteronyssinus, Dermato- phagoides farinae, Dermatophagoides siboney, Dermatophagoides microceaus, Blomia tropi- calis and Euroglyphus maynei, see for example, Thomas et al, 1998, Allergy 53, 821-832.
• Group 1 mite allergens share significant homology with a family of cysteine proteases including actinidin, papain, bromelain, ananain, cathepsin H, cathepsin K, and cathepsin B, which is why they often are referred to as Group 1 mite cysteine proteases.
• the Group 1 mite allergens are commonly found in the faeces of mites and are thought to function as digestive enzymes in the mite intestine.
• Group 1 allergens from different mites are highly homologous, approximately 25 kilo- dalton (kD) secretory glycoproteins, that are synthesized by the cell as a pre-pro-protein that is processed to a mature form. Maturation of this group of allergens have been reported e.g. by autocatalysis in which maturation of recombinant pro-Der p 1 was achieved by addition of the active natural Der p 1 cysteine protease (van Oort et al., 2002, Eur. J. Biochem. 269:671-679).
• the Group 1 allergens are very important in the production of allergy vaccines since dust mites are among the most common sources of allergy causing proteins which proteins include the Der p 1 and Der f 1 cysteine proteases.
• Der p 1 When Der p 1 was expressed in S. cere- visiae, the product was intracellular and insoluble (Chua et al., J. Allergy Clin. Immunol., 1992, vol. 89, 95-102). Even after solubilization and refolding the authors found clear antigenic differences from native protein.
• Der p 1 is expressed in other hosts, as e.g.
• the invention provides in a first aspect such an improved method for obtaining a cysteine protease in mature form comprising the steps: a) providing the cysteine protease propeptide from a eukaryotic host cell; and b) digesting the propeptide with a serine protease.
• the invention relates to a method for obtaining a Group 1 cysteine protease in mature form comprising the steps: a) providing the Group 1 cysteine protease propeptide from a host cell; and b) digesting the propeptide with a serine protease.
• the invention relates to a method for obtaining a Group 1 cysteine protease comprising expressing and secreting the Group 1 cysteine protease in Saccaromyces cerevisiae.
• Fig. 1 shows maturation of proDer p 1 protein. Crude broth containing protein of interest were incubated with BPN'. Proteins were analyzed in SDS-PAGE (A) and immunoblot analysis (B). Lane 1 : Marker, Lane 2: nDer p 1 , Lane 3: Filtron cone. proDer p 1 protein in crude broth pH 5 untreated sample, Lane 4: Maturation of proDer p 1 in Filtron crude broth, pH 5 after 4 hour incubation with BPN', Lane 5: Maturation of proDer p 1 in Filtron crude broth, pH 5 after 24 hours incubation with BPN', Lane 6: Filtron cone.
• proDer p 1 protein in crude broth pH 7 untreated sample Lane 7: Maturation of proDer p 1 in Filtron crude broth, pH 7 after 4 hours incubation with BPN'
• Lane 8 Maturation of proDer p 1 in Filtron crude broth, pH 5 after 21 hours incubation with BPN'.
• Fig. 2 shows maturation of proDer p 1 protein. Semi-purified (Phenyl Toyo Pearl) and purified (Q-Sepharose) proDer p 1 protein was incubated with BPN' in time interval. Proteins were analyzed in SDS-PAGE (A) and immunoblot analysis (B).
• Lane 1 Marker
• Lane 2 nDer p 1
• Lane 3 Semi-purified proDer p 1 , untreated sample
• Lane 4 Maturation of semi-purified proDer p 1 after 4 hours incubation with BPN'
• Lane 5 Maturation of semi-purified proDer p 1 after 24 hours incubation with BPN'
• Lane 6 Purified proDer p 1 , untreated sample
• Lane 7 Maturation of purified proDer p 1 after 4 hours incubation with BPN'
• Lane 8 Maturation of purified proDer p 1 after 4 hours incubation with BPN'.
• Fig. 3 shows maturation of proDer p 1 protein by B34.
• Fig. 4 shows maturation of proDer p 1 protein by BPN'.
• Purified proDer p 1 protein was incubated with BPN' in a time- and dose-range. Proteins were analyzed in an immunoblot analysis.
• Lane 1 Marker
• Lane 2 nDer p 1
• Lane 3 proDer p 1
• Lane 5 Maturated proDer p 1 incubated with 16.5 ⁇ g/ml BPN' 4 hours, pH 7
• Lane 6 Maturated proDer p 1 incubated with 16.5 ⁇ g/ml BPN' 24 hours, pH 7
• Lane 7 Maturated proDer p 1 incubated with 165 ⁇ g/ml BPN' 1 hour
• Lane 8 Maturated proDer p 1 incubated with 165 ⁇ g/ml BPN' 4 hours, pH 7, Lane 9
• Propeptides A propeptide coding region, codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
• the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
• a propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
• protease of the family of subtilisin-like serine proteases have shown useful for activation of propeptides.
• the propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factor gene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).
• proteases or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3).
• the polypeptide is provided as an inactive polypeptide including the propeptide from a host cell, and in a first aspect the polypeptide comprises cysteine proteases and the host cell is a Eukaryotic cell.
• Cysteine proteases means a protease defined as eukaryotic thiol proteases (EC: 3.4.22.-) (Dufour E. Sequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin. Biochimie 70: 1335- 1342 (1988)). This is a family of proteolytic enzymes which contain an active site cysteine. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby his- tidine side chain; an asparagine completes the essential catalytic triad. The order and spacing of these residues in the active sites vary in the 20 or so known families.
• Families 01 , C2 and C10 are loosely termed papain-like as the protein fold of the peptidase unit for members of this family resembles that of papain. Nearly half of all cysteine proteases are found exclusively in viruses (Rawlings N.D., Barrett A.J. Families of cysteine peptidases. Meth. Enzymol. 244: 461- 486 (1994)). Some of the proteins in this family are allergens. Allergies are hypersensitivity reactions of the immune system to specific substances called allergens (such as pollen, stings, drugs, or food) that, in most people, result in no symptoms.
• allergens such as pollen, stings, drugs, or food
• Cysteine proteases in the method of the present invention are obtained as a mature polypeptide in which the propeptide has been cleaved off.
• the mature protease in this context comprises both active forms of the mature protease as well as inactive forms of the protease which are allergenic. Allergenicity of a polypeptide indicates its ability to stimulate IgE antibody production and allergic sensitization in exposed animals, including humans.
• the cysteine protease comprises Group 1 mite allergens often referred to as Group 1 mite cysteine proteases.
• mites produce several classes or groups of allergens, one of which is known as Group 1 allergens.
• Group 1 allergens displaying considerable cross-reactivity, have been found in Dermatophagoides pteronyssinus, Dermatophagoides farinae, Dermatophagoides siboney, Dermatophagoides microceaus, Blomia tropi- calis and Euroglyphus maynei, see for example, Thomas et al, 1998, Allergy 53, 821-832.
• Group 1 mite allergens share significant homology with a family of cysteine proteases including aetinidin, papain, bromelain, ananain, cathepsin H, Cathepsin K, and cathepsin B (http://merops.sanqer.ac.uk/), which is why they often are referred to as Group 1 mite cysteine proteases.
• the Group 1 mite allergens are commonly found in the faeces of mites and are thought to function as digestive enzymes in the mite intestine.
• Group 1 allergens from different mites are highly homologous, approximately 25 kilo- dalton (kD) secretory glycoproteins, that are synthesized by the cell as a pre-pro-protein that is processed to a mature form.
• D. farinae, D. pteronyssinus, and E. maynei Group 1 proteins for example, share about 80% identity.
• Group 1 allergens from D. farinae and D. pteronyssinus also referred to as Der f 1 and Der p 1 proteins, respectively, show extensive cross-reactivity in binding IgE and IgG.
• Group 1 mite allergens thus include native polypeptides known in the art as Der p 1 obtainable from Dermatophagoides pteronyssinus (NCBI accession number: P08176, SEQ ID NO:1 in DK PA 2003 00628), Der f 1 obtainable from Dermatophagoides farinae (NCBI accession number: P16311 , SEQ ID NO:2 in DK PA 2003 00628), Eur m 1 obtainable from Eurogly- phus maynei (NCBI accession number: P25780, SEQ ID NO: 3 in DK PA 2003 00628), Der m 1 obtainable from Dermatophagoides microceaus (NCBI accession number: P16312, SEQ ID NO: 4 in DK PA 2003 ).
• group 1 mite allergens includes in particular native group 1 mite allergens, but also includes homologs to the native group 1 allergens, such as recombinant variants with disrupted N-glycosylation motifs, and hybrids of the above mentioned mite allergens, e.g. as created by family shuffling as described in the art (J.E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893-896, 1999).
• the polypeptide comprises Group 1 mite cysteine proteases and the host cell is any suitable host cell.
• the Group 1 mite cysteine proteases comprise in a particular embodiment Der p 1 , Der f 1 , and Der m 1.
• the cysteine protease according to the invention is digested with a serine protease in the step (b) of the method of the invention.
• Serine proteases In this context "serine protease” (EC 3.4.21.-) is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 "Principles of Biochemistry ,” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
• the bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropyl fluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease.
• alkaline protease covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 1 1.0 (for review, see Priest (1977) Bacteriological Rev. 41 71 1-753).
• Subtilases Siezen et al have proposed a sub-group of the serine proteases tentatively designated subtilases, Protein Engng, 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501- 523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases.
• subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997).
• subtilases I-S1 or "true” subtilisins
• subtilisin 168 subtilisin 168
• BASBPN subtilisin BPN'
• BLSCAR subtilisin Carlsberg
• I-S2 or high alkaline subtilisins is recognized by Siezen et al. (supra).
• Subgroup I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL ® , Gist-Brocades NV), subtilisin 309 (BLSAVI)(SAVINASE ® , NOVOZYMES A/S), subtilisin 147 (BLS147) (ESPERASE ® , NOVOZYMES A/S), and alkaline elastase YaB (BSEYAB).
• the serine protease comprises subtilisin (EC 3.4.21.14).
• suitable subtilisins comprises BPN' (for further description of the
• BPN' sequence see fig. 1 or Siezen et al., Protein Engng. 4 (1991 ) 719-737), Savinase (SAV- INASE ® , NOVOZYMES A/S), PD498 (Subtilisin from a Bacillus sp., GeneSeqP:AAW24071 ; W09324623A1) and B34 (Subtilisin from Bacillus alcalophilus; WO 0158275).
• Host cells The present invention also relates to recombinant host cells, comprising a nueleotide sequence or nueleotide construct or recombinant expression vector of the invention, which are advantageously used in the recombinant production of the cysteine proteases of the invention.
• the term "host cell” encompasses a parent host cell and any progeny thereof, which is not identical to the parent host cell due to mutations that occur during replication.
• the host cell is preferably transformed with a vector comprising a nueleotide sequence encoding the cysteine protease followed by integration of the vector into the host chromosome.
• Transformation means introducing a vector comprising a nueleotide sequence encoding the cysteine protease into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nueleotide sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non-homologous recombination as described above. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
• the host cell may in a particular embodiment be a unicellular mi- croorganism, e.g., a prokaryote, or a another particular embodiment a non-unicellular microorganism, e.g., a eukaryote.
• Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amylo- liquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Strep- tomyces murinus, or gram negative bacteria such as E.
• a Bacillus cell e.g., Bacillus alkalophilus, Bacillus amylo- liquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacill
• the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell.
• the transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizin, 1961 , Journal of Bacteriology 81 :823-829, or Dubnar and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56:209-221 ), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5771-5278).
• the host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.
• useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection. Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650 and 1651 ), BHK (ATCC CRL 1632, 10314 and 1573, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61 ) cell lines.
• the host cell is a fungal cell.
• Fungi as used herein in- eludes the phyla Aseomyeota, Basidiomyeota, Chytridiomycota, and Zygomyeota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171 ) and all mitosporic fungi (Hawksworth et al., 1995, supra).
• Examples of Basidiomyeota include mushrooms, rusts, and smuts.
• Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.
• Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya.
• mitosporic fungi examples include Aspergillus, Penicil- lium, Candida, and Alternaria.
• Representative groups of Zygomyeota include, e.g., Rhizopus and Mucor.
• the fungal host cell is a yeast cell.
• yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae.
• the latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saeeharomyces).
• the basidiosporogenous yeasts include the genera Leucospo dim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella.
• yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980.
• yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J.S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saeeharomyces, Strathern et al., editors, 1981 ).
• the yeast host cell may be selected from a cell of a species of Candida, Kluyveromyces, Saeeharomyces, Schizosaccharomyces, Candida, Pichia, Hansehula, , or Yarrowia.
• the yeast host cell is a Saeeharomyces carlsbergensis, Saccharomy- ces cerevisiae, Saeeharomyces diastatieus, Saeeharomyces douglasii, Saeeharomyces kluy- veri, Saeeharomyces norbensis or Saeeharomyces oviformis cell.
• yeast host cells are a Kluyveromyces lactis Kluyveromyces fragilis Hansehula polymorpha, Pichia pastoris Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279 and US 4,879,231 ).
• the fungal host cell is a filamentous fungal cell.
• “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
• the filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligated aerobic. In contrast, vegetative growth by yeasts such as Saeeharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
• the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myeeliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma or a teleomorph or synonym thereof.
• the filamentous fungal host cell is an Aspergillus cell.
• the filamentous fungal host cell is an Acremonium cell.
• the filamentous fungal host cell is a Fusarium cell.
• the filamentous fungal host cell is a Humicola cell.
• the filamentous fungal host cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal host cell is a Myeeliophthora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal host cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Trichoderma cell.
• the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger, Aspergillus nidulans or Aspergillus oryzae cell.
• the filamentous fungal host cell is a Fusarium cell of the section Discolor (also known as the section Fusarium).
• the filamentous fungal parent cell may be a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, or Fusarium trichothecioides cell.
• Fusarium bactridioides Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarc
• the filamentous fungal parent cell is a Fusarium strain of the section Elegans, e.g., Fusarium ox- ysporum.
• the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell.
• the filamentous fungal host cell is a Mucor miehei cell.
• the filamentous fungal host cell is a Myeeliophthora thermophilum cell.
• the filamentous fungal host cell is a Neurospora crassa cell.
• the filamentous fungal host cell is a Penicillium purpurogenum cell.
• the filamentous fungal host cell is a Thielavia terrestris cell or an Acremonium chrysogenum cell.
• the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cell.
• Aspergillus spp. for the expression of pro- teins is described in, e.g., EP 272 277, EP 230 023.
• the nueleotide sequences, encoding the cysteine protease of the invention may be modified such as to optimize the codon usage for a preferred particular host organism in which it will be expressed.
• the host cell is an insect cell and/or insect cell line.
• the insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). Culture conditions may suitably be as described in, for instance, WO 89/01029 or WO 89/01028, or any of the aforementioned references.
• the cysteine protease according to the invention may be expressed as a recombinant propolypeptide. Techniques for cloning and expression of recombinant polypeptides in different host organisms are well known in the art.
• nueleotide constructs Preparation of nueleotide constructs, vectors, host cells, protein variants and polymers for conjugation
• conventional molecular biology, microbiology, and recombinant DNA techniques well known to a person skilled in the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritseh & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989") DNA Cloning: A Practical Approach, Volumes I and II /D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed.
• the method may in a particular embodiment be carried out to express group 1 dust mite proteins as inclusion bodies in E.coli or in soluble form in methylotrophic yeasts such as Pichia pastoris, as described in WO 01/29078 (HESKA) describing recombinant expression of group 1 mite proteins including nueleotide sequences modified to enable expression of the polypeptides in microorganisms.
• Another particular method is to express group 1 dust mite proteins in insect cells such as Drosophila (Jacquet et al, Clin Exp. Allergy, 2000, vol. 30 pp.
• the polypetide variants of the invention may be prepared by (a) transforming a suitable host cell with a nueleotide construct capable of expressing the cysteine pprotease of the invention, (b) cultivating the recombinant host cell of the invention comprising a nueleotide construct of the invention under conditions conducive for production of the cysteine protease of the invention and (c) recovering the cysteine protease.
• the method may in a particular embodiment be carried out as described in WO 01/29078 (HESKA) describing recombinant expression of group 1 mite proteins including nueleotide sequences modified to enable expression of the polypeptides in microorganisms.
• HESKA WO 01/29078
• Transformation Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81 :1470-1474. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147- 156 or in copending US Serial No. 08/269,449. Examples of other fungal cells are cells of filamentous fungi, e.g., Aspergillus spp., Neurospora spp., Fusarium spp.
• 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 and EP 230 023.
• the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989, Gene 78: 147-156.
• Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
• Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
• Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in US 4,745,051 ; US 4, 775, 624; US 4,879,236; US 5,155,037; US 5,162,222; EP 397,485) all of which are incorporated herein by reference.
• the transformed or transfected host cells described above are cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.
• 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. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection). The media are prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
• the polypeptide variant of the invention is in an isolated and purified form.
• the polypeptide variant of the invention is provided in a preparation which is more than 20 %w/w pure, particularly more than 50% w/w pure, more particularly more than 75% w/w pure, more particularly more than 90% w/w pure and even more particularly more than 95% w/w pure.
• the purity in this context is to be understood as the amount of polypeptide variant of the invention present in the preparation of the total polypeptide material in the prepa- ration.
• the term "isolated" indicates that the polypeptide is found in a condition other than its native environment, such as apart from blood and animal tissue.
• the isolated polypeptide is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure. If the molecules are secreted into the nutrient medium, they can be recovered directly from the medium. If they are not secreted, they can be recovered from cell lysates. The molecules are 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.
• a salt e.g., ammonium sulphate.
• the proteins may be matured in vitro e.g., by acidification to induce autocatalytic processing (Jac- quet et al., Clin Exp Allergy, 2002, vol. 32 pp 1048-53), and they may be purified by a variety of chromatographic procedures, e.g., ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of molecule in question (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
• chromatographic procedures e.g., ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of molecule in question (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
• the cysteine protease propolypeptide provided, e.g. by culturing a recombinant host cell expressing the protease as described above, is according to the present invention di- gested in step (b) with a serine protease as defined above.
• the digestion can be performed in any suitable way known to the skilled person. In one particular embodiment of the invention the digestion is performed by co-expression of the serine protease in the host cell. In another embodiment the digestion is performed in vitro. In still another embodiment the serine protease is present in the culture broth during cultivation or added after cultivation.
• In vitro digestion of the cysteine protease propolypeptide can be performed on a crude extract comprising the propolypeptide or on isolated propolypeptide in different degrees of purity as defined above. During digestion the pH is adjusted to be in the range from pH 4-10, particularly from pH 5-9, more particularly from pH 7-8. After digestion of the propolypeptide the activity of the resulting mature cysteine protease can be determined by active site titration using a fluorescent substrate as exemplified below. In this respect it is advantageous to remove or inactivate the serine protease (e.g. subtilisin) first. In one embodiment of the invention this is done by adding a serine protease inhibitor after completing step (b).
• a serine protease inhibitor after completing step (b).
• the serine protease inhibitor is Ci-2A (Barley chymotrypsin inhibitor CI-2A).
• Ci-2A Barley chymotrypsin inhibitor CI-2A
• the CI-2A chymotrypsin inhibitor encoding gene of barley and the plasmid carrying the gene translated through an alfa leader sequence were described in US patent No. 5,674,833.
• the protein can thus be expressed and purified for use (see for example: Longstaff, C, Campbell, A.F., and Fersht, A.R. (1990) "Recombinant chy- motrypsin inhibitor 2: Expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism". Biochemistry, 29(31 ):7339-7347).
• Example 1 Construction and Expression of Der p 1
• the cysteine protease encoding gene of the present invention was located in vector pSteD212, which is derived from yeast expression vector pYES 2.0 (Invitrogen, UK and Kofod et al. 1994 J. Biol. Chem. 269: 29182-29189). This plasmid replicated both in E. coli and in S. cerevisiae. In S. cerevisiae Der p 1 was expressed from this plasmid.
• pSteD212 is an episomal expression vector containing URA3, gene of the synthetic pathway for uracil, encoding oritidine 5'-decarboxylase which allows for selection on minimal medium.
• the vector further contains 2 my Origin, origin of DNA replication to ensure multicopy of the plasmid in both yeast and E. coli.
• the TPI (triose-phosphate isomerase) promoter ensures constitutive expression of the gene of interest which can be cloned into a multiple cloning site (mcs) placed downstream of the promoter.
• mcs multiple cloning site
• a yeast transcriptional terminator is present downstream of the mcs.
• the ampieillin resistance gene also carried on pSteD212 is used for selection in E. coli.
• Example 2 Fermentation Fermentations for the production of Der p 1 enzyme was performed at 30 °C on a rotary shaking table (250 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml SC medium for 4 days. Consequently, in order to make e.g. a 2 litre broth 20 Erlenmeyer flasks were fermented simultaneously. Small scale fermentations in 10 ml SC medium in 50 ml sterile plastic tubes were also used.
• SC Medium (per litre): Yeast Nitrogen Base without amino acids 7.5 g Succinic acid 11.3 g Casamino acid without vitamine 5.6 g Tryptophan 0.1 g Add H 2 0. Autoclave and cool before adding glucose and L-threonin to a final concentration of 4 % and 0.02 %, respectively. For agar plates, 20 g bactoagar was added to the medium before autoclave.
• Example 3 Expression of Der p 1 protein
• the transformation solution was plated on SC-agar plates for colony formation at 30 °C, 3 days. Colonies were inoculated in 50 ml sterile plastic tubes, each tube containing 10 mL SC medium. The tubes were fermented at 30 °C, 250 r.p.m. for 4 days. Culture broth from these fermentations were used for sandwich ELISA experiments to determined the concentration of expressed protein.
• Sandwich ELISA Immunoplates (Nunc Maxisorb; Nunc-Nalgene) were coated overnight at 4 °C with at suitable dose of polyclonal rabbit anti Der p 1 antibody. The plates were then washed thoroughly with 0.15 M Phosphate Buffered Saline (PBS) containing 0.05 % Tween 20 (PBST), and remaining binding sites are blocked with PBS with 2 % skim milk powder, 1 h at room temperature. Samples, whether it is purified, semi-purified recombinant group 1 mite propolypeptide allergen or crude culture broth containing propolypeptide of interest, were added in a suitable dose or dose-range.
• PBS Phosphate Buffered Saline
• PBST 0.05 % Tween 20
• the plates were then washed thoroughly with 0.15 M PBST before the allergens were detected by incubation with biotinylated monoclonal anti Der p 1 antibody (INDOOR) 1 h at room temperature. Wash again in 0.15 M PBST. Conjugate with com- plexes of Streptavidin:Horse Radish Peroxidase (Kierkegaard & Perry) for 1 h at room temperature. Repeat washing step and develop by adding 3,3',5,5'-tetramethylbenzidine hydrogen peroxide (TMB Plus, Kem-En-Tec) and stop reaction by addition of 0.2 M H2S04.
• TMB Plus 3,3',5,5'-tetramethylbenzidine hydrogen peroxide
• OD450 reflected allergen binding to the immunoglobin, and by including natural Der p 1 (available from Indoor biotechnologies, NA-DP1 ) in known concentrations in the experiment in a dose rage the amount of Group 1 dust mite variant allergen bound could be determined.
• Example 4 Assay for detection and purification of propolypeptide form and mature form of Der p 1 antigen
• Method for fast qualitative detection of Der p 1 or pro Der p 1 was as follows. Immunosorp microtiter plates were bought from NUNC and microtiter wells were coated with 100 microlitres of 10 microgram per ml unlabelled polyclonal antibodies against Der P1 for overnight at 4 degree C. The microtiter wells were then washed with PBS Tween buffer as described in literature. Microtiter wells were then saturated with 200 microlitres of PBS buffer containing 10 milligrams per millilitres BSA and 0.05 % Tween 20 and incubated for 30 min- utes at room temperature. Microtiter wells were washed thrice with PBS buffer containing 0.05 % Tween 20.
• Microtiter wells were then coated with 100 microlitres fractions containing Der p 1 or pro-Der p 1 and incubated for 20 minutes with gentle shaking. Microtiter wells were then washed thrice with PBS buffer containing 0.05 % Tween 20. Microtiter wells were then coated with 100 microlitres of biotin labelled polyclonal antibodies around 1 microgram per millilitres diluted in PBS buffer with 0.05 % Tween 20 and incubated for 20 minutes at room temperature with gentle shaking. Microtiter wells were again washed thrice with PBS buffer and coated with 100 micro- litres of properly diluted Immunopure Avidin Horse radish peroxidase conjugate which was purchased from Pierce chemicals.
• Bound proteins were then eluted with linear gradient using buffer B containing 50 mM Borate pH 8 with 1 M salt as Sodium chloride. Total buffer used was 20 column volumes All the fractions were then analyzed by SDS-PAGE and qualitative ELISA assay.
• Ci-2A inhibitor was added in two Molar excess compared to BPN' and incubated at RT.
• BPN' can be separated from mature Der p 1 using hydrophobic chromatography with a phenyl sepharose column: The sample containing BPN' and mature Der p 1 is added ammonium sulphate to a final concentration of 1 M and applied onto the column. BPN' is eluted using a linear ammonium sulphate gradient from 1 to 0 M over 10 column volumes in 50 mM sodium phosphate, pH 7.0. Subsequently, mature Der p 1 is lib- erated from the column using a small volume of 20% ethanol.
• Maturated Der p 1 and proDer p 1 protein were detected with polyclonal rabbit-anti-Der p 1 antibody (DakoCytoma- tion) diluted 1 :2000 in TNT buffer (25 mM Tris-HCL, 0.5 M NaCl, 0.1% Triton x-100).
• Antigen- antibody complexes were detected with biotinylated goat-anti-rabbit Ig antibody (DakoCytoma- tion) diluted 1 :5000 in TNT buffer, and Horse-Radish Peroxidase (HRP)-conjugated Strepta- vidin (KPL, Maryland, USA) diluted 1 :2500.
• Membranes were washed 3 times in TNT buffer. Blots were developed using DAB (Sigma-Aldrich).
• Activity of maturated Der p 1 pro- tein was measured by mixing 25 ⁇ l sample with 25 ⁇ l Ci-2A inhibitor, 50 ⁇ l assay buffer (50 mM sodium phosphate, 1 mM EDTA, 0.0225% Brij 35, 20 mM cysteine, pH 7) and 100 ⁇ l substrate solution (60 ⁇ M N-Succinyl-Leu-Leu-Val-Tyr 7-Amido-4-Methylcoumarin in assay buffer) in the wells of a black flat-bottom microtiter plate. Liberation of the fluorophore over time was measured with excitation at 350 nm and emission at 460 nm on a spectrofluorometer instru- ment (Table 1 ). Alternatively, protein samples were analyzed on a SDS-polyacrylamide gel and in an immunoblot analysis as described.
• Table 1 Concentration ( ⁇ M) of Der p 1 after maturation of filtron concentrated crude proDer p 1 culture supernatant with BPN'. Concentrations are determined by comparing measured val- ues for Der p 1 with measured activity of commercial native Der p 1 (InDoor Technologies, NA- DP1 ) of known concentration determined by active site titration with E-64.
• Lane 2 The proform is hardly discemable and is at least 10x smaller than mature form.
• Lane 3 The proform is larger than the mature form, probably 2x as large. Lane 4: The proform and mature forms are about equal.
• Lane 5 The proform is smaller than the mature form, probably 1 ,5 x smaller.
• Lane 6 The proform is much (at least 5x) larger than the mature form (which is hardly discemable).
• Lane 7 The proform and the mature form are about equal. Lane 8: The proform is very weak and at least 5x smaller than the mature form.
• Activity of maturated Der p 1 protein was measured by mixing 25 ⁇ l sample with 25 ⁇ l Ci- 2A inhibitor, 50 ⁇ l assay buffer (50 mM sodium phosphate, 1 mM EDTA, 0.0225% Brij 35, 20 mM cysteine, ph 7) and 100 ⁇ l substrate solution (60 ⁇ M N-Succinyl-Leu-Leu-Val-Tyr 7-Amido- 4-Methylcoumarin in assay buffer) in the wells of a black flat-bottom microtiter plate. Liberation of the fluorophore over time was measured with excitation at 350 nm and emission at 460 nm on a spectrofluorometer instrument (Table 2). Alternatively, protein samples were analyzed in a SDS-polyacrylamide gel and in an immunoblot analysis as described.
• Lane 2 The proform is hardly discemable and is at least 10x smaller than mature form.
• Lane 3 The proform is much larger than the the mature form, probably at least 5x as large.
• Lane 4 The proform is much (about 5x) smaller than the mature form.
• Lane 5 The proform is not discemable and thus at least 10x smaller than the mature form.
• Lane 6 The proform is much (at least 5x) larger than the mature form (which is hardly discern- able).
• Lane 7 The proform is not discemable and the mature form is only a very weak band.
• the pro- form must be at least 2x smaller than the mature form based on the estimation that a band 2x weaker than the mature form would still be discemable.
• Lane 8 The proform is not discemable and the mature form is stronger than seen on lane 7.
• the proform must be at least 5x smaller than the mature form based on the estimation that a band 5x weaker than the mature form would still be discemable.
• subtilisin time- and dose-dependent maturation of proDer p 1 protein A time- and dose-dependent maturation of purified pro-Der p 1 protein by the addition of high (165 ⁇ g/ml) or low (16.5 ⁇ g/ml) concentration of subtilisins (BPN' or B34) at pH 7 or pH 8 was performed. After 1 h, 4h and 24h incubation with the subtilisins, the inhibitor, Ci-2A was added in two Molar excess to the subtilisins as described.
• Activity of maturated Der p 1 protein was measured by mixing 25 ⁇ l sample diluted 5 to 9 times in assay buffer (50 mM sodium phosphate, 1 mM EDTA, 0.0225% Brij 35, 20 mM cysteine, pH 7) with 25 ⁇ l Ci-2A inhibitor, 50 ⁇ l assay buffer and 100 ⁇ l substrate solution (60 ⁇ M N-Succinyl-Leu-Leu-Val-Tyr 7-Amido-4- Methylcoumarin in assay buffer) in the wells of a black flat-bottom microtiter plate. Liberation of the fluorophore over time was measured with excitation at 350 nm and emission at 460 nm on a spectrofluorometer instrument, (table 3). Alternatively, protein samples were analyzed in an immunoblot analysis as described.
• Table 3 Maturation of proDer p 1 with BPN' and B34. % activated calculated from activity of reference sample of commercial native Der p 1 (InDoor Technologies, NA-DP1 ) with known concentration determined by active site titration with E-64.
• Subtilisin B34 Maturation of pro-Der p 1 by the addition of low concentration of B34 (16.5 ⁇ g/ml), pH 7 only showed weak induction of time-dependent maturation. This was in contrast to pro-Der p 1 maturated by the addition of high concentration of B34 (165 ⁇ g/ml) that showed a time-dependent induction of maturated Der p 1 protein with the highest yield of maturated Der p 1 protien found at 24 hours incubation, pH 7. Analysis of maturation of samples at pH 8 also showed a time-dependent induction of maturation, however after 24 hours incubation, no visible bands were detected, probably due to digestion by the subtilisins in addition to low stability of the maturated Der p 1 protein.
• Subtilisin BPN' Induction of maturation of pro-Der p 1 protein by the addition of low concentration of BPN' (16.5 ⁇ g/ml), pH 7 showed a time-dependent increase of maturated Der p 1 protein (highest yield of maturated Der p 1 protein was reached by a 24 hour incubation). Induction of maturated protein also showed a dose-dependent increase and in samples incubated with high concentration of BPN' fully maturated protein was achieved by a 4 hour incu- bation.
• CI-2A corresponding to 5 to 50 times molar excess compared to added subtilisins
• CI-2A inhibits the activity of the subtilisins but not mature Der p 1.
• activity of maturated Der p 1 was measured by adding 75 ⁇ l substrate solution (60 ⁇ M N-succinyl-Leu-Leu-Val-Tyr 7-amido-4-methyl-coumarin in assay buffer) and release of fluorescent group was measured with excitation at 355 nm and emission at 460 nm.
• N-terminal sequencing by Edman degradation also gave the N-terminal: TNACSIN, demonstrating, that the site-specific processing by subtilisin works well for several different variants of Der p 1.
• Example 7 Maturation of pro Der p 1 with various non-subtilisin proteases: Maturation of recombinant pro-Der p 1 was attempted with seven non-subtilisin proteases: ALP (An Achromobacter lyticus protease. Swissprot:P15636. Lysyl endopeptidase. Peptidase clan SA - peptidase family S5), a Fusarium protease (trypsin-like protease from Fusa- hum oxysporum.
• Peptidase clan MA - peptidase family M36 C-component (from Bacillus licheniformis. Swissprot:P80057. Glutamyl endopeptidase. Peptidase clan SA - peptidase family S2B), Neutrase (Bacillolysin from Bacillus amyloliquefaciens. Gene- Seq:AAY44621. Novozymes commercial product. Swissprot:P06832.
• BPN' Subtil- isin Novo from Bacillus amyloliquefaciens. Peptidase clan SB - peptidase family S8. These proteases were added to final concentrations of 12.5, 25, 50, 100 and 200 ⁇ g/ml in 50 mM sodium phosphate at pH 7.0 with 25 ⁇ g/ml pro-Der p 1.
• activity of maturated Der p 1 was measured by mixing 10 ⁇ l sample with 65 ⁇ l assay buffer (50 mM sodium phosphate, 1 mM EDTA, 0.025% Brij 35, 20 mM cysteine, pH 7.0), 75 ⁇ l CI-2A (40 ⁇ M in assay buffer) and 75 ⁇ l substrate solution (0.05 mg/ml N-succinyl-Leu-Leu- Val-Tyr 7-amido-4-methyl-coumarin in assay buffer) in the wells of a black microtiter plate.
• Cl- 2A was added to inhibit activity of BPN', 10R and C-component on the fluorescently labelled substrate.
• Example 8 pH stability of mature Der p 1
• the stability of mature Der p 1 was tested by dissolving commercial native Der p 1 (from InDoor Technologies, NA-DP1 ) (12.5, 25, 50, 100 and 200 nM) in 50 mM sodium phosphate, 1 mM EDTA, 20 mM L-cysteine, 0.0225 % Brij 35 adjusted to various pH.
• activity was measured by mixing 50 ⁇ l sample diluted at least 10 times in assay buffer (50 mM sodium phosphate, 1 mM EDTA, 20 mM L-cysteine, 0.0225% Brij 35, pH 6.0) with 50 ⁇ l substrate solution (60 ⁇ M N-succinyl-Leu-Leu-Val-Tyr 7- amido-4-methyl-coumarin in assay buffer) in the well of a microtiter plate. Fluorescence with excitation at 355 nm and emission at 460 nm was measured every minute for 20 minutes.

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