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WO1996016177A1 - Procede de production de polypeptides a allergenicite reduite - Google Patents

Procede de production de polypeptides a allergenicite reduite Download PDF

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Publication number
WO1996016177A1
WO1996016177A1 PCT/DK1995/000463 DK9500463W WO9616177A1 WO 1996016177 A1 WO1996016177 A1 WO 1996016177A1 DK 9500463 W DK9500463 W DK 9500463W WO 9616177 A1 WO9616177 A1 WO 9616177A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
zipper
kda
polypeptides
dna
Prior art date
Application number
PCT/DK1995/000463
Other languages
English (en)
Inventor
Mads Eskelund BJØRNVAD
Annette PRENTØ
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to JP8516467A priority Critical patent/JPH10509324A/ja
Priority to EP95936995A priority patent/EP0793726A1/fr
Priority to AU39240/95A priority patent/AU3924095A/en
Publication of WO1996016177A1 publication Critical patent/WO1996016177A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/70—Biological properties of the composition as a whole
    • A61K2800/72—Hypo-allergenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a process for producing polypeptides with reduced allergenicity. Further to a DNA construct comprising genes encoding such polypeptides, a recombinant expression vector or transformation vehicle comprising said DNA construct, a cell harbouring said DNA construct or vector. Also contemplated is polypeptides with reduced allergenicity produced according to the process of the invention, oligomeric polypeptides with reduced allergenicity, and compositions comprising said polypeptides. Finally the invention relates to the use of Zipper domains for reducing the allergenicity of polypeptides.
  • polypeptides including enzymes and non-enzymatic proteins, are being produced industrially for use in industry, household, food/feed, cosmetics, medicine etc. Being polypeptides they are capable of stimulating the immune systems of animals and humans.
  • risk groups include employees handling the manufacturing products comprising enzymes and professions such as hairdressers which are daily in direct contact with products comprising polypeptides.
  • certain polypeptides can elicit the production of different kinds of antibodies and/or give a cellular response. At least one of these routes can give adverse effects in humans and animals, as exposure to polypeptides may result in sensitisation and subsequently allergy. While sensitisation is defined as an immune status, allergy is characterized as a clinical disease. Allergy in general requires two or more encounters with antigens. The first exposure lead to a primary immune response which results in sensitisation of the individual. If the sensitised individual comes in contact with the same antigen again it may provoke an allergic response.
  • IgE attach to specific receptors on the surface of mast cells, which contain many large cytoplasmic granules packed with chemical mediators. Once attached to a mast cell, the IgE molecule can survive for many weeks with its antibody reaction site, available to interact with a specific allergen. Individuals having IgE-mediated allergy have many IgE antibodies fixed to mast cells. Upon exposure the specific allergen molecules readily combines with the cell-fixed corresponding IgE antibodies. This leads to cellular release of the cytoplasmic granules of chemical mediators, which gives symptoms like rhinitis, conjunctivitis, uricaria or other allergic reactions.
  • IgE-mediated hypersensitivity reaction may occur when the allergen is introduced via the respiratory tract by inhalation.
  • allergic responses The occurrence of allergic responses is believed at least partly to depend on the way of exposure. For instance, it have been found that intranasal challenge with allergenic proteins provokes an allergic response even though skin tests and radioallergosorbent test (RAST) for specific serum IgE are negative (Ivan Roitt, "Essential Immunology", fifth edition, p. 152 and p. 240, 1984).
  • RAST radioallergosorbent test
  • prior art methods for reducing the allergenicity of polypeptides consist of various ways of immobilizing, granulating, coating or dissolving the polypeptides to avoid especially polypeptides in dust form from stimulating the immune system.
  • polypeptides of human origin for production, e.g. in bacteria or mammalian cell cultures. This may alleviate some problems for humans, but not for animals. Furthermore, it will in many cases not be possible to find polypeptides of human origin with the desired properties, for which reason other origin has to be considered. This can be either human polypeptides that are altered in one or more positions in the molecule, giving performance that is desired. It might also be molecules from other species, including bacteria, mold etc. All the latter groups of products will have potency for immune stimulation.
  • Such a conjugate may also exhibit novel properties: e.g. EP 38 154 (Beecham Group Ltd.) discloses conjugates of allergens with polysarcosine which have immunosuppressive properties.
  • US patent no. 4,179,337 (Enzon) concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. Between 10 and 100 moles of polymer are used per mole polypeptide and at least 15% of the physiological activity is maintained. The protected polypeptide is injected in an aqueous solution either into the mammalian circulatory system or intramuscularly. The non-immunogenicity is assessed from intradermal injection tests.
  • EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful polypeptides linked to at least one water-soluble polymer by means of a reversible linking group.
  • EP 471 125 (Kanebo, LTD.) describes a modified protease linked to a polysaccharide via a triazine ring leading to a suppressing effect on antigenicity and dermal hypersensitivity.
  • the employed polysaccharide has an average molecular weight not less than 10,000.
  • the modification rate for surface amino acid groups in the modified protease is not less that 30%.
  • allergens entering the respiratory tract, must have a molecular weight lower than about 100 kDa in order to penetrate the plasma membrane and cause allergic reactions.
  • WO 94/10191 discloses a process for production of low allergenic protein, wherein the monomeric parent protein molecules are linked together to form an oligomer. This is done e.g. by using a linker or spacer molecule or by linking the monomeric molecules together by peptide bonds between the C-terminal of the first monomer and the N-terminal of the second monomer.
  • EP 215 662 (Masda, Hiroshi) concerns a modified or unmodified protease derived from microorganisms for use in medicaments such as anti-tumour agent.
  • the modification of the protease may be carried out by forming dimers or oligomers by cross-linking the protease molecules.
  • Zero technique is known to cause oligomerization of polypeptides.
  • Zipper domains include the well-known ⁇ -helical bundles, crossed bundles, multiple bundles, parallel coiled coils, poly(L-glutamine) strands.
  • the simplest cases of Zipper domains include the helical bundles which consist of amphiphilic helices, e.g. Leucine Zippers and four ⁇ -helical bundles. These domains share a characteristic seven-amino acid repeat of the type (a,b,c,d,e,f,g) n . Positions "a” and “d” of the heptad repeat are generally hydrophobic, a property which signals the potential for the interlocking of ⁇ -helices, as suggested by Crick (Acta crystallogr., 6, p. 689-697, 1953). Despite the common pattern different sequences form two-, three, four-stranded and even higher order stranded helical bundles (Cohen et.
  • Leucine Zipper is the 33 amino acid sequence located at the C-terminus of GCN4, a yeast transcription factor, which belongs to a class of DNA binding polypeptides (O'Shea et al. Science, 243, p. 538-542, 1989).
  • GCN4 Leucine Zipper Through genetic engineering the specific GCN4 Leucine Zipper has been fused to different polypeptides and shown to mediate dimerization of monomeric polypeptides.
  • Leucine Zippers form homo-dimers, but within the group of Leucine Zippers there are specific motifs which favour the formation of hetero-dimers.
  • Two examples of such are the Fos and Jun Leucine Zipper (O'Shea et al. Science, 245, p. 646, 1989; Turner and Tjian, Science, 243 p. 1689, 1989) and the artificially made hetero dimeric coiled coil described by O'Shea (Current Biology, vol. 3, no 10, p. 658-667, 1993).
  • Another polypeptide motif which can mediate trimerization is the naturally occurring motif of the shock transcription factor of Saccharomyces cerevisiae and Kluveromyces lactis described by Peteranderl et al. Biochemistry, 31, p. 12272-12276, 1992.
  • Examples of tetrameric formation involves altering amino acid residues in the GCN4 Leucine Zipper (Harbury et al., Science, 262, p. 1401-1407, 1993. This was only shown for small peptides.
  • An oligomerization motif from myosin fused to recombinant protein can mediate the formation of higher order polypeptide oligomers (Wolber et al., BIO/TECHNOLOGY, 10, p. 900-904, 1992).
  • the expressed fusion polypeptide forms oligomers at low salt concentration and dissociates at high salt concentrations.
  • the present inventors have perceived the potential of using Zipper domains for industrial uses, and have now surprisingly accomplished to provide a process for producing a polypeptide with reduced allergenicity, by
  • said microorganism has been modified by the introduction of one or more DNA constructs comprising a DNA sequence coding for at least one polypeptide and at least one Zipper domain.
  • Another object of the invention is to provide a DNA construct for the production of polypeptides with reduced allergenicity, comprising a DNA sequence encoding at least one polypeptide molecule with at least one Zipper domain.
  • the invention also relates to a recombinant vector or transformation vehicle, comprising said DNA construct of the invention, and furthermore to a cell comprising said DNA construct or said recombinant vector or transformation vehicle.
  • compositions comprising at least one polypeptide component of the invention.
  • the invention relates to the use of Zipper domains for reducing the allergenicity of polypeptides.
  • Figure 1 shows the DNA sequence and derived amino acid sequence of a linker, the GCN4 Leucine Zipper and a flexible C-terminal extension peptide containing a cystein amino acid residue.
  • Figure 2 shows the construction steps of the pAZ-1 plasmid.
  • Figure 3 shows a coomassie blue stained SDS-PAGE run under nonreducing conditions, wherein lane 4 is a molecular weight marker SeeBlueTM (Cat. :#LC5625, Novex, Inc., Ca, USA), and lanes 2 and 3 are polypeptides expressed by E. coli JM105 comprising the DNA construct pAZ-1.
  • SeeBlueTM Cat. :#LC5625, Novex, Inc., Ca, USA
  • Figure 4 shows coomassie blue stained SDS-PAGE run under reducing conditions, wherein lane l is a molecular weight marker SeeBlueTM (Cat.:#LC5625, Novex, Inc., Ca, USA), and lanes 2 and 3 are polypeptides expressed by E. coli JM105 comprising the DNA construct pAZ-1.
  • SeeBlueTM Cat.:#LC5625, Novex, Inc., Ca, USA
  • lanes 2 and 3 are polypeptides expressed by E. coli JM105 comprising the DNA construct pAZ-1.
  • Figure 5 shows a Western blot.
  • Lane 1 is the molecular weight marker.
  • Lanes 2 and 3 are samples from the induced JM105/pAZ-1 run under non-reduced conditions.
  • Lanes 4 and 5 are the same samples run under reduced conditions.
  • Figure 6 shows the number of Dunkin Hartley guinea pig, having been exposed to 1.0 ⁇ g monomer and 1.0 ⁇ g dimer Termamyl ® intratracheally, found to be IgG 1 positive vs. days starting from the day of exposure.
  • the present inventors have now surprisingly succeeded in providing an integrated industrially applicable process for producing polypeptides with reduced allergenicity, wherein the biological activity of the polypeptides is at least substantially maintained.
  • a “substantially” maintained activity is in the context of the present invention defined as an activity which is at least between 20% and 30%, preferably between 30% and 40%, more preferably between 40% and 60%, better from 60% up to 80%, even better from 80% up to about 100%, in comparison to the activity of the not modified parent polypeptide.
  • Said polypeptides may be used for a vast number of industrial applications which will be exemplified below.
  • the term “reduced allergenicity” indicates that the amount of produced IgE (in humans, and molecules with comparable effects in specific animals, for instance IgG, in guinea pigs), which can lead to an allergic state are significantly decreased when inhalating a polypeptide of the invention in comparison to the corresponding parent polypeptide.
  • immunogen immunogen
  • antigen antigen-derived neuropeptide
  • an “immunogen” may be defined as a substance which when in- troduced into humans and animals is capable of stimulating an immunologic response.
  • antigen refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule by the immune system.
  • an "allergen” may be defined as an antigen which may give rise to allergic sensitization or an allergic response by IgE antibodies (in humans, and molecules with comparable effects in animals).
  • allergen is the wider term and includes "antigen” and "allergen”.
  • allergen is the wider term and includes "antigen” and "allergen”.
  • epidermatitis As mentioned above it is, at least in the context of polypeptides of the present invention, important to distinguish between dermal allergens mediating allergic responses caused by skin contact, and respiratory allergens causing allergic responses by contact with cell-bound IgE in the bronchial tree, due to the well-known fact that skin tests may be negative even though inhalation tests provoke an allergic response.
  • allergenicity may be made by inhalation tests, comparing the effect of intratracheal administrated parent polypeptides with the corresponding polypeptides of the invention with reduced allergenicity.
  • Animal models recommended by ECETOC includes both mice and guinea pig models.
  • mice models focus upon events occurring during the induction phase of sensitisation following primary encounter of the substance in question. However, mice are not considered suitable for investigating polypeptides.
  • guinea pig models seek to identify respiratory allergens as a function of elicitation reactions induced in previously sensitised animals.
  • ECETOC assesses results of studies using guinea pig as a suitable basis for hazard assessment in man.
  • models involving introduction of polypeptides intratracheal in guiniea pigs are suitable.
  • One suitable strain of guinea pigs the Dunkin Hartley strain, does not (as humans) produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgG,A and IgG,B which characterize their allergenic response to inhaled polypeptides (see e.g. Prent ⁇ , ATLA, 19, p. 8-14, 1991).
  • the relative amounts of IgG,A and IgG,B are a measure for the allergenicity level.
  • polypeptide with reduced allergenicity specifically comprises
  • the allergenicity of the polypeptides is believed to be reduced by the enlargement of the polypeptides.
  • self-oligomerization does in the context of the present invention mean joining together a desired number of polypeptide molecules, e.g. by the use of Zipper domains, and includes dimerization, trinterization, tetramerization, multimerization, polymerization etc.
  • the microorganism is modified by introducing one or more DNA construct (s) into said microorganism.
  • Said DNA construct comprises a DNA sequence encoding at least one polypeptide of interest operably linked to at least one Zipper domain.
  • the DNA sequence may further comprise a short linker sequence between the sequence encoding the polypeptide and the Zippper domain and/or a DNA sequence encoding a purification tag.
  • the recovery of the oligomerized polypeptide may be carried out in any suitable way.
  • the polypeptides may be recovered by IMAC (Immobbilized Metal Affinity Chromatography) following the procedure described in e.g. Yip et al., (1994), Molecular Biotechnology, vol. 1, p. 151-164; Fatiadi et al., (1987), CRC Critical Rev. Anal. Chem. 18, p. 1-44.
  • a linker sequence is a DNA sequence encoding an amino acid sequence connecting the polypeptide in question and the Zipper domain.
  • the enlargement of the polypeptides takes place during fermentation.
  • the amino acid sequence of the Zipper domain is expressed grafted to the N- or C-terminal of the polypeptide of interest.
  • Zipper domains associate e.g. two and two and are held together by hydrophobic and electrostatic interactions.
  • the fusion polypeptide may also form trinters, tetramers etc.
  • the process of the invention is advantageous due to the fact that no additional step need to be executed after the fermentation and before the recovery to obtain the polypeptide product with reduced allergenicity.
  • process according to the invention may be used for any polypeptides of interest, which may be any polypeptides that in parent form may cause an allergic reaction.
  • This group comprises polypeptides having a molecular weight below about 100 kDa.
  • the said molecular weight lies in the range of between about 5 kDa and 150 kDa, preferably from between about 20 kDa and 100 kDa, especially from between about 20 kDa and 80 kDa.
  • polypeptides may be of microbial or mammalian origin and may be naturally occurring polypeptides or variants thereof.
  • polypeptide of interest is an enzyme exhibiting at least one catalytic activity.
  • Such enzymes may be selected from the group comprising proteases (metallo, acid, neutral or alkaline), lipases, cutinases, cellulases, amylases, lyases, xylanases, pectinases, pullulanase, polygalacturonases, oxidases, laccases, oxidoreductases, transglutaminases, ⁇ -galactpcidases, phytases and peroxidases
  • proteases metalo, acid, neutral or alkaline
  • lipases cutinases
  • cellulases amylases
  • lyases lyases
  • xylanases pectinases
  • pullulanase polygalacturonases
  • oxidases laccases
  • oxidoreductases transglutaminases
  • ⁇ -galactpcidases phytases and peroxidases
  • Termamyl ® Novo Nordisk
  • trimeric, tetrameric, multimeric polypeptides and/or enzymes exhibiting one or more catalytic activities are also contemplated.
  • the polypeptide with reduced allergenicity may be produced by any suitable bacteria or fungal organisms as described below.
  • Another object of the invention is to provide a DNA construct for the production of polypeptides with reduced allergenicity, comprising a DNA sequence encoding at least one polypeptide, and at least one Zipper domain.
  • DNA construct is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA, RNA or PNA origin.
  • construct is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a DNA sequence encoding a polypeptide of interest fused to at least one Zipper domain.
  • the construct may optionally contain other DNA segments, such as a short linker sequence and/or a sequence encoding a peptide segment specifically used for purification purposes.
  • the DNA construct 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 parts of the polypeptide of interest by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY, 1989).
  • the DNA sequence may encode a polypeptide exhibiting catalytic activities.
  • the DNA sequence may encoding at least one enzyme selected from the group comprising proteases (metallo, acid, neutral or alkaline), lipases, cutinases, cellulases, amylases, lyases, xylanases, pectinases, polygalacturonases, oxidases, laccases, oxidoreductases, transglutaminases, ⁇ -galactosidases, phytases or peroxidases.
  • proteases metalo, acid, neutral or alkaline
  • lipases cutinases
  • cellulases cellulases
  • amylases lyases
  • xylanases xylanases
  • pectinases polygalacturonases
  • oxidases laccases
  • oxidoreductases transglutaminases
  • ⁇ -galactosidases
  • the DNA construct of the invention may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters, 22, p. 1859 - 1869, 1981, or the method described by Matthes et al., EMBO Journal, 3, p. 801 - 805, 1984.
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • the DNA 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 DNA construct, in accordance with standard techniques.
  • the DNA construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science, 239, p. 487 - 491, 1988.
  • the DNA construct of the invention comprises the DNA sequence shown in SEQ ID NO 1 as well as nucleic acid sequences encoding the amino acid sequence shown in SEQ ID NO 2, but may differ from the DNA sequence shown in SEQ ID NO 1 by virtue of the degeneracy of the genetic code.
  • the present invention relates to a recombinant vector or transformation vehicle 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.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is in- dependent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the polypeptide of interest to be self-oligomerized is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • 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 of interest.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding polypeptides either homologous or heterologous to the host cell.
  • promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem., 255, p. 12073 - 12080, 1980; Alber and Kawasaki, J. Mol. Appl. Gen., 1, p. 419 - 434, 1982) 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 TPI1 (US 4,599,311) or ADH2-4c (Russell et al., Nature, 304, p. 652 - 654, 1983) promoters.
  • suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. , 4, p. 2093 - 2099, 1985) or the tpiA promoter.
  • suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • Preferred are the TAKA-amylase and gluA promoters.
  • 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 pumilus xylosidase gene, or by the phage Lambda P R or P L promoters or the E. coli lac, trp or tac promoters.
  • the DNA sequence may also, if necessary, be operably connected to a suitable terminator.
  • 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, p. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, phleomycin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • Schizosaccharomyces pombe TPI gene described by P.R. Russell, Gene 40, 1985, p. 125-130
  • a drug e.g. ampicillin, kanamycin, tetracyclin, phleomycin, chloramphenicol, neomycin, hygro
  • selectable markers include amdS, pyrG, argB, niaD, trpC and sC.
  • a secretory signal sequence also known as a leader sequence, prepro sequence or pre sequence
  • 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 polypeptide.
  • 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 ⁇ -factor signal peptide (cf. US 4,870,008), the signal peptide of mouse salivary amylase (cf. 0. Hagenbuchle et al., Nature, 289, p. 643-646, 1981), a modified carboxypeptidase signal peptide (cf. L.A. Vails et al.. Cell, 48, p.
  • yeast BAR1 signal peptide cf. WO 87/02670
  • yeast aspartic protease 3 YAP3
  • 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 ⁇ -factor leader (the use of which is described in e.g.
  • 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 or WO 92/11378.
  • the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.
  • the signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral ⁇ -amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
  • said vector is the pAZ-1 expression vector.
  • the DNA sequence encoding the fusion polypeptide in question, 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 secretory 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.
  • 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 polypeptide of interest and includes bacteria, yeast, filamentous fungi.
  • bacterial host cells which, on cultivation, are capable of producing the polypeptide of interest are grampositive bacteria such as strains of Bacillus, such as strains of B . subtilis, B . licheniformis, B. lentus, B . brevis, B . stearothermophilus, B . alkalophilus, B . amyloliquefaciens , B . coagulans , B . circulans, B . lautus, B. megaterium or B.
  • 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).
  • the polypeptide 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.
  • sonication or osmotic shock to release the contents of the periplasmic space and recovering the polypeptide.
  • 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 polypeptide 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.
  • suitable yeast cells are strains of Kluyveromyces , such as K. lactis , Hansenula, e .g. H. polymorpha, or Pichia, e .g. P. pastor is (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, p. 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 polypeptides is described in, e .g. , EP 272 277, EP 238 023 and EP 184 438.
  • the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al.. Gene, 78, p. 147-156, 1989.
  • a filamentous fungus When 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 host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the polypeptide of interest, 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. 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 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.
  • a salt e.g. ammonium sulphate
  • the invention is also directed towards polypeptides with reduced allergenicity produced according to a process of the invention.
  • the polypeptide of the invention consist of a Zipper domain fused to the polypeptide of interest.
  • the N- or C-terminal of the amino acid sequence of the polypeptide is grafted to the Zipper domain.
  • the Zipper domain may be any domain capable of oligomerizing the polypeptides in the production phase.
  • the Zipper domain is a Leucine Zipper, such as the GCN4 Leucine Zipper.
  • the monomeric polypeptide has a molecular weight of between 5 kDa and 150 kDa, preferable between 20 kDa and 100 kDa, especially between 20 kDa and 80 kDa.
  • Leucine Zippers When using Leucine Zippers for oligomerizing polypeptides consisting of two polypeptide molecules, such as two Termamyl ® molecules, the Leucine Zippers normally have enough affinity to keep the homo-dimer stable. However, to further stabilize the dimer, a cysteine may be included in the Leucine Zipper. This construction can lead to the formation of a disulfide bridge between the two monomers of hybrid polypeptide.
  • the polypeptide of the invention comprises from 2 to 10 polypeptide molecules or more.
  • the self-oligomerized polypeptide is a dimer, a trimer, a tetramer, or an oligomer.
  • polypeptide of the invention may exhibit more than one biological activity, e.g. two or more different enzymatic activities, such as lipolytic and proteolytic activities.
  • the invention also related to oligomeric polypeptides with reduced allergenicity comprising at least one polypeptide bonded or linked to at least one Zipper domain which is coupled to at least one polypeptide bonded or linked to at least one Zipper domain.
  • Said oligomeric polypeptides may be homo-oligomeric, hetero-oligomeric or higher order oligomeric polypeptide produced by any suitable process or prepared by any suitable method.
  • Said Zipper domain may be any of the previously mentioned Zipper domains.
  • said oligomeric polypeptide exhibits at least one of the previously mentioned enzymatic activities.
  • Said Zipper domain may be linked to either the C- or N-terminal of the polypeptide(s) in question.
  • polypeptide includes both polypeptides produced according to the process of the invention and said oligomeric polypeptides of the invention.
  • a polypeptide according to the invention may demonstrate a high degree of controlled stability.
  • polypeptides may advantageously be irreversible fused together, which entails that the product has only negligible tendency to disintegrate, which would lead to the return of conditions that may cause an allergenic state.
  • polypeptides stay oligomerized in the production and/or bulk handling phase, but dissociates later on, when the polypeptides does not inflict a risk of exposure to humans or animals.
  • the cleavage of the linkage between the polypeptides may be activated e.g. by physical conditions, such as pH, ionic strength, temperature, reduction or oxidation potential etc.
  • the invention also relates to a composition comprising at least one polypeptide and/or at least one oligomeric polypeptide of the invention.
  • composition may further comprise other ingredients normally used in e.g. detergents, including soap bars, household articles, agrochemicals, personal care products, cosmetics, toiletry, pharmaceuticals, composition used for treating textiles, food and/or feed etc.
  • detergents including soap bars, household articles, agrochemicals, personal care products, cosmetics, toiletry, pharmaceuticals, composition used for treating textiles, food and/or feed etc.
  • Detergent compositions including soap bars, household articles, agrochemicals, personal care products, cosmetics, toiletry, pharmaceuticals, composition used for treating textiles, food and/or feed etc.
  • a polypeptide of the invention may be an enzyme used in detergent compositions. It may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG poly(ethylene oxide) products
  • PEG polyethyleneglycol
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition may be in any convenient form, e.g. as powder, granules, paste or liquid.
  • a liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or nonaqueous.
  • the detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic.
  • the detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap.
  • anionic surfactant such as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap.
  • nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).
  • AEO or AE alcohol ethoxylate
  • carboxylated alcohol ethoxylates such as carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).
  • the detergent composition may additionally comprise one or more enzymes, such as e.g. amylases, lipases, cutinases, proteases, cellulases, peroxidases, and oxidases.
  • enzymes such as e.g. amylases, lipases, cutinases, proteases, cellulases, peroxidases, and oxidases.
  • the detergent may contain 1-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylene-diaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA) , alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may also be unbuilt, i.e. essentially free of detergent builder.
  • the detergent may comprise one or more polymers.
  • CMC carboxymethylcellulose
  • PVP poly(vinylpyrrolidone)
  • PEG polyethyleneglycol
  • PVA poly (vinyl alcohol)
  • polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent may contain a bleaching system which may comprise a H 2 O 2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS).
  • TAED tetraacetylethylenediamine
  • NOBS nonanoyloxybenzenesulfonate
  • the bleaching system may comprise peroxyacids of, e.g., the amide, imide, or sulfone type.
  • the detergent composition of the invention comprising the polypeptide of the invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as, e.g., an aromatic borate ester, and the composition may be formulated as described in, e .g. , WO 92/19709 and WO 92/19708.
  • stabilizing agents e.g. a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative such as, e.g., an aromatic borate ester
  • the detergent may also contain other conventional detergent ingredients such as, e .g. , fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil-redeposition agents, dyes, bactericide ⁇ , optical brighteners, or perfume.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil-redeposition agents, dyes, bactericide ⁇ , optical brighteners, or perfume.
  • the pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11.
  • the manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching". Nature, 369, p. 637-639, 1994.
  • Detergent composition formulated as a nonaqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali.
  • a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali.
  • the detergent may also comprise anionic surfactant and/or a bleach system.
  • the enzyme of interest of the invention may be incorporated in concentrations conventionally employed in detergents. It is at present contemplated that, in the detergent composition of the invention, the enzyme in question with reduced allergenicity may be added in an amount corresponding to 0.001-100 mg of enzyme per liter of wash liquor.
  • polypeptides of the invention with reduced allergenicity may also advantageously be used in dishwashing detergents.
  • Dishwashing detergent compositions comprise a surfactant which may be anionic, non-ionic, cationic, amphoteric or a mixture of these types.
  • the detergent will contain 0-90% of non-ionic surfactant such as low- to non-foaming ethoxylated propoxylated straight-chain alcohols.
  • the detergent composition may contain detergent builder salts of inorganic and/or organic types.
  • the detergent builders may be subdivided into phosphorus-containing and non-phosphorus-containing types.
  • the detergent composition usually contains 1-90% of detergent builders.
  • Examples of phosphorus-containing inorganic alkaline detergent builders when present, include the water-soluble salts especially alkali metal pyrophosphates, orthophosphates, and polyphosphates.
  • suitable organic builders include the alkali metal, ammonium and substituted ammonium, citrates, succinates, malonates, fatty acid sulphonates, carboxymetoxy succinates, ammonium polyacetates, carboxylates, polycarboxylates, amino- polycarboxylates, polyacetyl carboxylates and polyhydroxysulphonates .
  • suitable organic builders include the higher molecular weight polymers and co-polymers known to have builder properties, for example appropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acid copolymers and their salts.
  • the dishwashing detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type.
  • inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite and hypobromite as well as chlorinated trisodium phosphate.
  • organic chlorine/bromine-type bleaches are heterocyclic N-bromo and Nchloro imides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water-solubilizing cations such as potassium and sodium.
  • Hydantoin compounds are also suitable.
  • oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound.
  • suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates and perphosphates.
  • Preferred activator materials are TAED and glycerol triacetate.
  • the dishwashing detergent composition of the invention may be stabilized using conventional stabilizing agents for the enzyme (s), e.g. a polyol such as e.g. propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester.
  • a polyol such as e.g. propylene glycol
  • a sugar or a sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester.
  • the dishwashing detergent composition of the invention may also contain other conventional detergent ingredients, e.g. deflocculant material, filler material, foam depressors, anti-corrosion agents, soil-suspending agents, sequestering agents, antisoil redeposition agents, dehydrating agents, dyes, bacteri cides, fluorescers, thickeners and perfumes.
  • polypetide of the invention may be used in conventional dishwashing detergents, e.g. in any of the detergents described in any of the following patent publications:
  • EP 346137 US 5112518, EP 318204, EP 318279, EP 271155,
  • polypeptides, including enzymes, of the invention with reduced allergenicity may be used in applications for purposes in the textile industry involving handling of enzyme granulates or powders.
  • Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appearance without loss of fabric wettability. Bio-polishing may be obtained by applying the method described e.g. in WO 93/20278. iii. Desizing
  • polypeptides according to the invention are of interest. In the following are listed examples of uses.
  • Proteases are well-known active ingredients for cleaning of contact lenses. They hydrolyse the proteinaceous soil on the lens and thereby makes it soluble. Removal of the protein soil is essential for the wearing comfort.
  • Proteases are also effective ingredients in skin cleaning products, where they remove the upper layer of dead keratinaseous skin cells and thereby makes the skin look brighter and more fresh.
  • Proteases are also used in oral care products, especially for cleaning of dentures, but also in dentifrices.
  • Lipases can be applied for cosmetic use as active ingredients in skin cleaning products and anti-acne products for removal of excessive skin lipids, and in creams and lotions as active ingredients for skin care. Lipases can also be used in hair cleaning products (e.g. shampoos) for effective removal of sebum and other fatty material from the surface of hair.
  • hair cleaning products e.g. shampoos
  • Lipases are also effective ingredients in products for cleaning of contact lenses, where they remove lipid deposits from the lens surface.
  • oxidase usually glucoseoxidase
  • substrate e.g. glucose
  • peroxidase usually lactoperoxidase
  • Antimicrobial systems comprising the combination of an oxidase and a peroxidase are know in the cleaning of contact lenses.
  • oxidoreductases are the application of oxidases, peroxidases and laccases in oxidative hair dyeing.
  • the free radicals activate chain reactions that lead to destruction of fatty membranes, collagen, and cells.
  • Protein disulfide isomerase is also an oxidoreductase. It can be utilised for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds).
  • Plaque formed on the surface of teeth are composed mainly of polysaccharides. They stick to the surface of the teeth and the microorganisms.
  • the polysaccharides are mainly ⁇ -1,6 bound glucose (dextran) and ⁇ -1,3 bound glucose (mutan).
  • glucanases such as mutanase and dextranase helps hydrolysing the sticky matrix of plaque, making it easier to remove by mechanical action.
  • other kinds of biofilm for instance the biofilm formed in lens cases can be removed by the action of glucanases.
  • Antimicrobial polypeptides have widespread applications such as preservation of cosmetic products, anti-acne products, deodorants and shampoos.
  • polypeptides with reduced allergenicity may further advantageously be used in food- and feedstuff.
  • Specifically relevant polypeptides are enzymes selected from the group of proteases, ⁇ -glucanases, amylases, pectinases, ⁇ -galactosidases, phytases, xylanases and lipases.
  • Use of Zipper domains are enzymes selected from the group of proteases, ⁇ -glucanases, amylases, pectinases, ⁇ -galactosidases, phytases, xylanases and lipases.
  • the invention relates to the use of Zipper.domains for reducing allergenicity of polypeptides and may be any molecules capable of self-oligomerizing microbially expressed polypeptides.
  • the Zipper domain is a Leucine Zipper.
  • the Leucine Zipper may be any known Leucine Zipper which, grafted to a polypeptide, is capable of self-oligomerizing as a result of association of parallel ⁇ -helical coils of two or more Leucine Zippers.
  • Leucine Zipper is the yeast transcriptional factor GCN4 or modifications thereof.
  • the Zipper domain may advantageously be a Fos Leucine Zipper and a Jun Leucine Zipper.
  • the Zipper domain is a four helical bundle or a modification thereof.
  • the Zipper domains may advantageously be used for reducing allergenicity of polypeptides in detergents, household articles, agrochemicals, personal care products, cosmetics, toiletry, pharmaceuticals, composition used for treating textiles, food and feed etc.
  • polypeptides comprising at least one Zipper domain may advantageously be used in compositions and/or in context with e.g. the industrial applications previously described.
  • Escherichia coli JM105 (Yanisch-Perron et al. Gene, 33, p. 103-119, 1985)
  • Escherichia coli MC1061 (Casadaban, M.J. et al., J. Mol.Biol. 138, p. 179-207, 1980).
  • the pFab3 expression vector is the ancestor of pFab4 ( ⁇ rum, H. et al., Nucleic Acids Research, 21, p. 4491-4498, 1993).
  • the vector contains a pelB signal sequence (Lei et al., J. of Bacteriol, vol. 169, p. 4379-4383, 1987) which is under control of the inducible lacZ promoter.
  • a SfiI site in the pelB signal makes it possible to clone the desired sequence, so that the geneproduct will be expressed In Frame with the signal sequence.
  • pFab3 contains 131 bp region between the start codons of the lacZ and the pelB signal.
  • the underlined nucleotides correspond to the termamyl sequence.
  • Primer A-termamyl also includes the SfiI restriction site and the last two codons of the pelB signal.
  • Primer B-termamyl includes a linker sequence (a short hinge domain of IgG3) (PlUckthun, A. et al.. Biochemistry, 31, p. 1579-1584, 1992) and a Xmal cloning site.
  • ⁇ -amylase EPS assay (Cat. :#1442295, Boehringer Mannheim GmbH, Mannheim, Germany)
  • Termamyl ® (available from Novo Nordisk A/S)
  • T4-DNA ligase (Cat.:#M1801, Promega Corporation, WI, USA) AmpliTaq ® DNA Polymerase (PartNo.:N801-0060, Perkin Elmer, Roche Molecular Systems, New Jersey, USA.)
  • PCR reaction buffer dNTP(0.25mM of each), MgCl 2 2.5mM and 1 ⁇ PCR reaction buffer-II (PartNo.:N808-0009, Perkin Elmer, Roche Molecular Systems, New Jersey, USA.)
  • T4-DNA ligase buffer (Cat. :#M1801, Promega Corporation, WI, USA) SOC medium (Sambrook, J. et al., 1989, Molecular Cloning.
  • PEG-8000 (Cat.:#P2139, Sigma Chemical Company, MO, USA).
  • the pH is adjusted to 9.0 with HCl, and Milli-Q water is applied to 1 litre.
  • the pH is adjusted to 10 with about 22.5 g KOH in Milli-Q water to 1 litre.
  • Thermocycler Varius V 45 (Hans Landgraf, GmbH, Langenhagen, Germany.)
  • HiTrapTM chelating column (Code no. 17-0409-01, Pharmacia LKB, Biotechnology AB, Uppsala, Sweden)
  • the PCR reaction is performed in 50 ⁇ l volume PCR reaction-buffer, 1 ⁇ M of each primer A- and B-termamyl and 10 ng of the DNA template.
  • Reaction mixtures are overlaid with mineral oil and kept at 94°C for 5 minutes. Then 0.5 ⁇ l of AmpliTaq ® (5 U/ ⁇ l) is added. The mixtures are kept at annealing temperature 70°C for 5 minutes and at elongation temperature 72°C for 2 minutes.
  • the four oligonucleotides are synthesized on Applied Biosystems
  • oligonucleotides are purified using denaturing polyacrylamide gel electrophoresis according to (Ausubel, F.M. et al., supra, 1994). 20 pmol of each oligonucleotide are mixed in a total volume of 40 ⁇ l of 100 mM NaCl, annealed by incubation at 95°C for 5 minutes and cooled slowly to 16°C over a period of 3 hours, this annealing mixture is used in ligation.
  • the four oligonucleotides are as follows:
  • the transformation is carried out by electroporation.
  • Epicurian coli ⁇ XL1-Blue Electroporation competent Cells are used, 3 ⁇ l of ligated DNA is used per 80 ⁇ l of cells.
  • the electroporation is performed using a Bio-Rad E. coli pulser set at 25 ⁇ F, 2.5 kV and 200 Ohms.
  • Transformation of E. coli JM105 is done by adding 3 ⁇ l of the ligation mixture per 100 ⁇ l of heat-shock competent cells. The preparation and transformation of the cells are made essentially as described in (Sambrook et al. 1989, supra.)
  • Termamyl ® -dimer in E. coli JM105 is done as follows. An overnight culture of JM105 harbouring the pAZ-1 plasmid in 2XTY medium with 100 ⁇ g/ml Ampicillin and 1% D(+) Glucose is prepared by transferring a single colony to the media and incubating this at 37°C for 16 hours with vigorously shaking. 100 ⁇ l of this is used as starter culture of 100 ml 2XTY medium with 100 ⁇ g/ml Ampicillin and 0.1% D(+)-Glucose, which in a 1 L shake flask is incubated at 37°C with vigorously shaking.
  • the periplasmic fraction of induced and non-induced cells harbouring the pAZ-1 plasmid are analyzed on an SDS-page gel 4-20% Acrylamide (Laemmeli, Nature, 227, p. 680, 1970) using the Mini-Protean II (Bio-Rad Laboratories, Richmond, Ca, USA). Samples are run with or without reducing agent dithiothreitol. Dithiothreitol (DTT) also termed Clelands reagent is a reducing agent capable of quantitatively reducing disulfide bonds (Cleland, W.W., Biochemistry, 3, p. 480, 1964).
  • DTT dithiothreitol
  • Clelands reagent is a reducing agent capable of quantitatively reducing disulfide bonds (Cleland, W.W., Biochemistry, 3, p. 480, 1964).
  • the expressed Termamyl ® Zipper dimer was purified from the fermentation broth. This was done using the attached poly-His tail as the affinity tag. More specifically the purification was performed using a 5 ml HiTrapTM chelating column according to the recommodations of the supplier. Further details concerning the IMAC (Immobbilized Metal Affinity Chromatography) procedure is describe in Yip et al., (1994), Molecular Biotechnology, vol. 1, p. 151-164; Fatiadi et al., (1987), CRC Critical Rev. Anal. Chem. 18, p. 1-44.
  • Termamyl ® Zipper dimer sample After purification and dialysis of the Termamyl ® Zipper dimer sample, spectral measurements were performed. The optical density of the sample at 280 nm were used to calculate the protein concentration of the sample. For this purpose the Lambert Beer law was used together with the calculated protein extinction coefficient of the Termamyl ® Zipper dimer using the principals for this as described by Gill et al. Analytical Biochemistry, 182, p. 319-326, 1989.
  • ELISA procedure for determination of IgG 1 positive guinea pigs ELISA microtiter plates are coated with rabbit anti-Termamyl ® AAN 4080 K 452-453 1:4000 in carbonate buffer and incubated over night at 4°C. The next day the plates is blocked with 2% BSA for 1 hour and washes 3 times with PBS tween 20. Termamyl ® PPX 3328 1 ⁇ g enzyme protein/ml is applied to the plates, incubated for 1 hour and then washed 3 times with PBS tween 20. All guinea pig samples are applied to the ELISA plates with 25 ⁇ l sera and 25 ⁇ l PBS buffer, incubated for 3 hours and washed 3 times with PBS tween 20.
  • goat anti-guinea pig IgG (diluted to 1:4000 in PBS buffer) is applied to the plates, incubated for 1 hour and washed 3 times with PBS tween 20.
  • Alkaline phosphatase rabbit anti-goat is applied (diluted to 1:8000) and incubated for 1 hour, washed 2 times in PBS tween 20 and 1 time with diethanol amine buffer.
  • Positive and negative sera values are calculated as the avarage blind values added 2 times the standard deviation. This gives an accuracy of 95%.
  • EXAMPLE 1 PCR amplification and cloning of the gene encoding Termamyl ® .
  • the primers A-termamyl and B-termamyl were designed and synthesized on Applied Biosystems 394 DNA/RNA synthesizer.
  • the Termamyl ® encoding gene was PCR amplified using the plasmid pDN1528 containing the gene encoding Termamyl ® as a template.
  • the PCR product a 1.5 kb fragment, was purified by preparative agarose electrophoresis followed by the Geneclean-II procedure (BIO 101, Inc., Ca., USA).
  • the purified 1.5 kb DNA fragment containing the sequence encoding Termamyl ® was digested with 10 U of SfiI per ⁇ g DNA at 50°C for 2 hours with the reaction mixture overlaid by mineral oil. Following the Geneclean-II procedure the DNA fragment was further digested for 2 hours at 37°C with 10 U XmaI per ⁇ g DNA. The digested DNA was again purified using Geneclean-II procedures and finally ligated to the prepared SfiI and XmaI digested pFab3.
  • the 10 ⁇ l ligation mixture included 0.2 ⁇ g of insert DNA and 0.2 ⁇ g of digested vector pFab3.
  • the ligation was performed for 2 hours at 16°C and 14 hours at 4°C with 1 U of T4-DNA ligase.
  • the ligated material was used to transform Epicurian coli ⁇ XL1-Blue Electroporation competent Cells as described above.
  • the isolated plasmid DNA was analyzed by digestion with SfiI and XmaI and the digested plasmid DNA was analyzed on 1% agarose, 1 ⁇ TBE gel. The appearance of a DNA fragment of 1.5 kb indicated the existence of clones containing the correct fragment size. Further verification of the cloned gene was done as DNA sequencing using the fmolTM DNA-sequencing system from Promega Corporation. A construct with the correct DNA sequence of Termamyl ® was named pAZ-1 ⁇ 2. EXAMPLE 4
  • plasmid DNA minipreps were prepared according to (Sambrook et al., supra, 1989). The isolated plasmid DNA was analyzed by digestion with Nrul (introduced by the Leucine Zipper DNA fragment), the digested and non-digested plasmid DNA were analyzed on 1% agarose, IxTBE gel. The appearance of linearized plasmid of 4606 bp in Nrul digested samples indicated the existence of clones comprising the Leucine Zipper fragment. Further verification of the cloned fragment was done by sequ encing using the fmolTM DNA-sequencing system from Promega Corporation.
  • JM105 cells harbouring the plasmid pAZ-1 were induced to express the fusion polypeptide pelB signal-Termamyl ® -Linker-Leucine Zipper as described above. Each fusion polypeptide dimerize with other identical fusion polypeptides during expression. Cells were given osmotic shock in order to release proteins present in the periplasma. Aliquots of the isolate from both induced and non-induced cells were analyzed on SDS-PAGE. Samples were analyzed under reducing (sample with DTT) and non-reducing (sample without DTT) conditions. Polypeptide bands were visualised by staining with Coomassie Blue dye.
  • Non-reduced samples of induced cells showed a distinct band at approximately 120 kDa (see figure 3) a band not present in samples of non-induced cells.
  • Reduced samples from induced cells showed a distinct band at approximately 60 kDa, while at the same time no band was seen at 120 kDa (see figure 4). In samples of non-induced cells no 60 kDa band were seen.
  • a gel similar to the above were used to transfer the proteins to a PVDF membrane via a Western blot procedure, as described above.
  • the 120 kDa band of non-reduced samples was specifically recognized as Termamyl ® confirming the expression of Termamyl ® as a dimer (see figure 5).
  • a sample of the periplasmic isolate was analyzed. It was estimated by electrophoretic analysis that this sample contained about 0.5 mg/ml of Termamyl ® -dimer. Dilutions of the sample were tested in an ⁇ -amylase assay (see the Materials and Methods section) and compared to dilutions of Termamyl ® of known activity. This assay revealed that the dimer retained more than 50% of the wild-type activity.
  • a purification tag was introduced at the C-terminal part of the Termamyl ® -Zipper protein as an in-frame insertion between the Xma I and Sad sites of pAZ-1 ⁇ 2. This resulted in a nucleotide sequence encoding the Termamyl ® -Zipper protein with a C-terminal tail consisiting of a Factor Xa site and the amino acid sequence His-His-His (see sequence data). Four oligonucleotides were used for this purpose (See below).
  • the four oligonucleotides were synthesized on Applied Biosystems 394 DNA/RNA synthesizer according to protocols of the supplier. After synthesis, the oligonucleotides were purified using denaturing polyacrylamide gel electrophoresis according to (Ausubel, F.M. et al., supra. 1994).
  • each oligonucleotide 20 pmol was mixed with a total volume of 40 ⁇ l of 100 mM NaCl, annealed by incubation at 95°C for 5 minutes and cooled slowly to 16°C over a period of 3 hours. This annealing mixture was used for ligation.
  • the four oligonucleotides were as follows: Antisense Zip-Xa-His (XmaI-SacI) (SEQ ID NO 7)
  • P indicates oligonucleotides with a phosphoryl group at the 5'-ends.
  • the oligonucleotides were designed to have extruding overhangs when hybridized: one matching the Xmal site and one matching the Sad site of pAZ-1 ⁇ 2.
  • the preperation of the pAZ-1 ⁇ 2 for ligation with the hybridized oligonucleotides, the actual ligation and transformation of competent E. coli JM105, were performed essentially as described in example 4 and in the Methods and Materials-section. From this transformation positive clones were identified as described in Example 5 and again the DNA sequence was verified by DNA sequencing. Further, dimer Termamyl ® was expressed as described in Example 6.
  • Figure 6 shows the number of Dunkin Hartley guinea pigs found IgG, positive during the trail period. It can be seen from figure 6 the number of guinea pigs being IgG, positive at any time during the trial period is reduced for the dimer Termamyl ® in comparison to the monomer Termamyl ® . This proves that the allergenicity of Termamyl ® can be reduced by coupling Termamyl ® to a Zipper domain.

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Abstract

L'invention concerne un procédé de production de polypeptides à allergénicité réduite qui consiste a) à faire fermenter un micro-organisme capable de produire ledit polypeptide, b) à récupérer ledit polypeptide sous une forme sensiblement pure, ledit micro-organisme étant modifié de sorte que les molécules du polypeptide exprimé s'auto-oligomérisent. L'invention porte également sur un ADN de recombinaison comprenant les gènes codant lesdits polypeptides, sur un vecteur d'expression de recombinaison ou un véhicule de transformation comprenant ledit ADN de recombinaison, sur une cellule abritant ledit ADN ou vecteur. Elle se rapporte encore à des polypeptides produits par des microbes présentant une allergénicité réduite et obtenus selon le procédé décrit, et à des compositions comprenant lesdits polypeptides. Pour finir, elle concerne l'utilisation des domaines Zipper pour réduire l'allergénicité des polypeptides.
PCT/DK1995/000463 1994-11-24 1995-11-23 Procede de production de polypeptides a allergenicite reduite WO1996016177A1 (fr)

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JP8516467A JPH10509324A (ja) 1994-11-24 1995-11-23 抑制されたアレルゲン性を有するポリペプチドの製造のための方法
EP95936995A EP0793726A1 (fr) 1994-11-24 1995-11-23 Procede de production de polypeptides a allergenicite reduite
AU39240/95A AU3924095A (en) 1994-11-24 1995-11-23 A process for producing polypeptides with reduced allergenicity

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WO1998028411A2 (fr) * 1996-12-23 1998-07-02 Genencor International, Inc. Compositions a base de cellulase agrandie utilisables pour le traitement de textiles
WO1999053038A2 (fr) * 1998-04-15 1999-10-21 Genencor International, Inc. Proteines mutantes avec une reponse allergene plus faible chez l'homme et methodes pour construire, identifier, et produire ces proteines
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6586224B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
WO2003066847A2 (fr) 2002-02-08 2003-08-14 Novozymes A/S Variants de phytase
WO2004111224A1 (fr) 2003-06-19 2004-12-23 Novozymes A/S Proteases ameliorees et procedes de production de ces dernieres
WO2004111221A1 (fr) 2003-06-19 2004-12-23 Novozymes A/S Proteases
US6838269B1 (en) 1998-04-15 2005-01-04 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6897049B1 (en) 1998-04-15 2005-05-24 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
WO2006037327A2 (fr) 2004-10-04 2006-04-13 Novozymes A/S Polypeptides ayant une activite de phytase et polynucleotides codant ces polypeptides
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WO2007107573A1 (fr) 2006-03-22 2007-09-27 Novozymes A/S Utilisation de polypeptides a activite antimicrobienne
WO2007112739A1 (fr) 2006-04-04 2007-10-11 Novozymes A/S Variants de phytase
WO2008116878A1 (fr) 2007-03-26 2008-10-02 Novozymes A/S Phytase de hafnia
WO2010034835A2 (fr) 2008-09-26 2010-04-01 Novozymes A/S Variant de phytase de hafnia
EP2258839A1 (fr) 2004-06-21 2010-12-08 Novozymes A/S Protéases de Nocardiopsis
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EP2284259A2 (fr) 2003-10-10 2011-02-16 Novozymes A/S Protéases
EP2418214A1 (fr) 2006-12-22 2012-02-15 Novozymes A/S Utilisation de polypeptides contre les maladies causées par des protozoaires
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WO1994010191A1 (fr) * 1992-10-30 1994-05-11 Novo Nordisk A/S Proteines faiblement allergenes

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WO1994010191A1 (fr) * 1992-10-30 1994-05-11 Novo Nordisk A/S Proteines faiblement allergenes

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WO1998028411A3 (fr) * 1996-12-23 1998-10-22 Genencor Int Compositions a base de cellulase agrandie utilisables pour le traitement de textiles
WO1998028411A2 (fr) * 1996-12-23 1998-07-02 Genencor International, Inc. Compositions a base de cellulase agrandie utilisables pour le traitement de textiles
US5989899A (en) * 1996-12-23 1999-11-23 Genencor International, Inc. Oversized cellulase compositions for use in detergent compositions and in the treatment of textiles
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
US6835550B1 (en) 1998-04-15 2004-12-28 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
WO1999053038A2 (fr) * 1998-04-15 1999-10-21 Genencor International, Inc. Proteines mutantes avec une reponse allergene plus faible chez l'homme et methodes pour construire, identifier, et produire ces proteines
US6936249B1 (en) 1998-04-15 2005-08-30 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
WO1999053038A3 (fr) * 1998-04-15 2000-02-10 Genencor Int Proteines mutantes avec une reponse allergene plus faible chez l'homme et methodes pour construire, identifier, et produire ces proteines
US6596525B1 (en) 1998-04-15 2003-07-22 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
US6897049B1 (en) 1998-04-15 2005-05-24 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6838269B1 (en) 1998-04-15 2005-01-04 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
US6586224B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
WO2003066847A2 (fr) 2002-02-08 2003-08-14 Novozymes A/S Variants de phytase
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WO2008116878A1 (fr) 2007-03-26 2008-10-02 Novozymes A/S Phytase de hafnia
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EP2650364A1 (fr) 2008-09-26 2013-10-16 Novozymes A/S Variant de phytase de hafnia
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CN113151330A (zh) * 2021-03-30 2021-07-23 云南师范大学 一种酸性蛋白酶突变体及其制备方法和应用
CN113151330B (zh) * 2021-03-30 2023-09-08 云南师范大学 一种酸性蛋白酶突变体及其制备方法和应用

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