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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—Enzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• Peptides, and/or enzymes, having antimicrobial activity may also be capable of inhibiting the outgrowth of Escherichia coli (DSM 1576) for 24 hours at 25°C in a microbial growth substrate, when added in a concentration of 1000 ppm; preferably when added in a concentration of 500 ppm; more preferably when added in a concentration of 250 ppm; even more preferably when added in a concentration of 100 ppm; most preferably when added in a concentration of 50 ppm; and in particular when added in a concentration of 25 ppm.
• Peptides, and/or enzymes, having antimicrobial activity may be capable of reducing the number of living cells of Bacillus subtilis (ATCC 6633) to 1/100 after 30 min.
• SEQ ID NO: 47 is amino acids 1-50 of bovine lactoferrin (LFB(1-50), and lactoferricin B (abbreviated LFcinB, or LFB(17-41)) designates amino acids 17-41 of SEQ ID NO: 47.
• SEQ ID NOs: 48-52 viz. LFB(14-31), LFB(17-31), LFB(18-31), LFB(19-31), and
• LFB(20-31), respectively are additional lactoferrin fragments disclosed in J. Peptide Sci. 5: 32-45 (1999) by Rekdal et al, who also discloses SEQ ID NOs: 53-55, viz. variants LFB(17- 31)17K, LFB(17-31)20F, and LFB(17-31)17K+20F, respectively.
• Additional lactoferhcins e.g. SEQ ID NOs. 56-57, viz. LFB(17-30), and LFB(19-37), respectively, are disclosed by Groenink et al in FEMS Microbiology Letters 179 (1999) 217- 222. According to Vogel et al (Biochem. Cell. Biol.
• protease having amino acids 1-188 of SEQ ID NO: 59 in which Ala in position 87 is substituted with Thr.
• Other preferred proteases are the following: The protease derived from Nocardiopsis solonvillei subsp. josonvillei and comprising the amino acid sequence of the mature part (amino acids 1-188) of SEQ ID NO: 60. The protease derived from Nocardiopsis alba and comprising the amino acid sequence of the mature part (amino acids 1-188) of SEQ ID NO: 61. The protease derived from Nocardiopsis prasina and comprising the amino acid sequence of the mature part (amino acids 1-188) of SEQ ID NO: 62.
• the one or more added amino acid(s) is (are) non-polar or uncharged; ii) the one or more added amino acid(s) is one or more of Q, S, V, A, or P; iii) the one or more added amino acids are selected from the group consisting of: QSHVQSAP (SEQ ID NO: 84), QSAP (SEQ ID NO: 85), QP, TL, TT, QL, TP, LP, TI, IQ, QP, PI, LT, TQ, IT, QQ, and PQ.
• an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced.
• the cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the antimicrobial agent or the enzyme, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated.
• Modification of a nucleic acid sequence encoding an antimicrobial agent or an enzyme as defined in claim 1 may be necessary for the synthesis of variant agents or variant enzymes.
• control sequence is an appropriate promoter sequence, a nucleic acid sequence that is recognized by a host cell for expression of the nucleic acid sequence.
• the promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide.
• the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
• the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell.
• the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
• Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans those phosphate isomerase.
• the nucleic acid sequences encoding the enzyme(s) may be the same or different.
• the nucleic acid construct of the invention may also comprise more than one nucleic acid sequence encoding more than one antimicrobial agent, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty nucleic acid sequences encoding one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty antimicrobial agents.
• AMP Anti Microbial Peptide
• ENZ Enzyme
• RBS Ribosome Binding Site
• PROM Promoter
• TERM Terminator
• PROM-RBS1-Gene(AMP)-Linker-Gene(ENZ)-TERM b) PROM-RBS1 -Gene(AMP)-RBS2-Gene(ENZ)-TERM
• Linker designates a cleavable linker, which is described in more detail below, and which provides for subsequent cleavage of the fusion protein, AMP-Linker-ENZ, into distinct products, AMP, and ENZ, respectively.
• Ad b) This construct results in one transcriptional product, but two translational products, as the translation will stop because of the translational stop codon inherent in Gene(AMP). Accordingly, two distinct products, AMP and ENZ, will be produced.
• Ad c) In this construct, transcription will stop after Gene(AMP), but the transcription continues at PROM2. Accordingly, two transcription products, as well as two translational products, AMP and ENZ, result from this construct.
• a still further specific cleavage method is available for D-/-P (Asp-Pro) peptide bonds, which can be cleaved by treatment with a weak acid (e.g. pH 2-3) at a suitable temperature, for a suitable time period.
• a weak acid e.g. pH 2-3
• suitable temperatures are 40, 50, 60, 70, or 80°C
• suitable time periods are from a few minutes to a few hours, e.g. % hour to 5 hours, 1 / hour to 2 1 /2 hours, 1 hour to 3 hours, VA hours to 2A hours.
• Protection Peptides also relates to the use, in the recombinant expression of antimicrobial peptides, of a so-called quenching domain, or protection peptide, or protection domain, which is characterized in that at least 50% of its component amino acid residues are E and/or D.
• the purpose of such protection domain is to temporarily inactivate the antimicrobial peptide, in case it would be inhibiting the growth of the host cell. Once sufficient quantities of the peptide have been produced, the protection peptide can be cleaved off as described further below, thus re-activating the antimicrobial peptide.
• the protection peptide is preferably synthetic (artificial).
• the length of the protection peptide is described in more detail below.
• the protection peptide and the antimicrobial peptide may be separated by a stretch of amino acids of up to 5, up to 8, up to 10, up to 12, up to 15, up to 20, up to 25, or up to 30 amino acids.
• the nucleic acid sequence encoding the peptide protection domain may be upstream or downstream of the antimicrobial peptide encoding nucleic acid sequence.
• 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). 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).
• filamentous fungal host cells are cells of species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.
• 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.
• the antimicrobial agent may be detected using antibodies directed against one or more epitopes on the peptide, or the peptide may be detected using antimicrobial activity.
• the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot) or engineered variants thereof.
• fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.
• water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate.
• Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.
• Examples of macro minerals are calcium, phosphorus and sodium.
• Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma- linoleic acid.
• Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
• An animal feed composition according to the invention has a crude protein content of
• the animal feed composition of the invention contains at least one vegetable protein or protein source as defined above. It may also contain animal protein, such as Meat and Bone Meal, and/or Fish Meal, typically in an amount of 0-25%.
• a recombinant host cell comprising at least one nucleic acid construct that comprises i) a first nucleic acid sequence encoding a peptide, said peptide a) encompassing no more than 100 amino acids, and b) having antimicrobial activity; and ii) a second nucleic acid sequence encoding an enzyme; preferably the recombinant host cell comprises a first nucleic acid construct, and a second nucleic acid construct, wherein i) the first nucleic acid construct comprises the first nucleic acid sequence encoding the peptide; and ii) the second nucleic acid construct comprises the second nucleic acid sequence encoding the enzyme; more preferably the recombinant host cell comprises a nucleic acid construct comprising i)
• DNA sequence encoding a member of the library of peptide protection candidate and a second DNA sequence encoding an antimicrobial peptide c) transforming host cells with the library of DNA constructs of b) and cultivating the transformed host cells to obtain expression of the DNA constructs; d) estimating, and/or analyzing, viability of the transformed host cells, and/or estimating, and/or analyzing, the yield of antimicrobial peptide; and e) identifying peptide protection candidates which when used in a DNA construct according to step b), for transformation of a host cell according to step c), results in an increased viability of the host cell, and/or an increased yield of antimicrobial peptide, when estimated, and/or analyzed, according to step d); as well as a protection peptide obtainable, and/or obtained, by such method.
• pHH1535 (DDDDE-PR39) Primer pHH1535-Forw: catagcaccatggacgatgacgatgaaaggagacgtccccgacccccatatttgcc (SEQ ID NO: 70)
• the added quenching domain can be liberated from the AMP allowing for monitoring of the antimicrobial activity of the AMP.
• the PR39-derivatives were amplified using specific Forward primers and a general reverse primer pBAD-rev that are indicated below in a standard PCR reaction.
• the PCR templates used were those generated in Example 3.
• the expression cassette was an amyQ promoter from Bacillus amyloliquefaciens in front of the crylllA promoter plus the mRNA stabilization element from Bacillus thuringiensis. (see US patent no 6,255,076, in particular the pDG268 neo system shown in Fig. 19 thereof).
• the following DNA fragments were prepared: (A) The Shine Delgarno sequence (SD) and the signal peptide were derived from the amyL gene of Bacillus licheniformis (ATCC 14580), viz.
• the plasmid with the above expression cassette and the cat gene flanked by amyE sequences was then transferred to competent cells of Bacillus subtilis WB600.
• a colony with the correct expression cassette inserted into the amyE gene was isolated, and the gene sequence of the inserted pectate lyase fusion peptide confirmed by DNA sequencing.
• the pectate lyase gene is fused in frame to the DNA sequence encoding the AFP from Aspergillus giganteus.

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• 2004
  • 2004-09-13 ES ES04762825T patent/ES2368221T3/es not_active Expired - Lifetime
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  • 2004-09-13 DK DK04762825.0T patent/DK1680503T3/da active
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