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  • 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)
  • C12N9/2462—Lysozyme (3.2.1.17)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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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
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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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
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
• • 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
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Lysozyme-analogous proteins and peptides with antimicrobial activity their production and their use
• the present invention relates to lysozyme-analogous proteins and peptides with antimicrobial activity including antiviral activity, in particular T4 lysozyme-analogous proteins and peptides with antimicrobial activity, their production and their use.
• the fields of application for this invention range from human and veterinary medicine to resistance breeding in plants and the preventive use of bactericidal and fungicidal additives in foods.
• T4 lysozyme is a protein that is produced by bacteriophage T4 in order to open the bacterial host cell after it has multiplied and to get into the environment. So far it has been assumed that the enzyme activity (muramidase) of the T4 lysozyme kills the bacteria (Tsugita, A. (1971) Phage lysozyme and other lytic enzymes; in: Boyer, PD (ed.) The Enzymes, Vol. V, Academic Press, New York, pp. 344-411). This requires transport through the bacterial cell membrane to the murein layer, which is the substrate of the T4 lysozyme.
• the previously known enzymatic activity of the T4 lysozyme leads to a specific cleavage between the C-1 of the muramic acid group and the C-4 of acetylglucosamine. This breaks down the bacterial muramine network and destabilizes the cell wall.
• the route of transport of the T4 lysozyme through the inner cell membrane to the muramine layer has not yet been clarified. As a result of this cracking of the muramine layer, bursting of the bacteria through destabilization and the associated killing was assumed.
• lysozyme genes are understood to mean any nucleic acid (DNA) which, after their transcription in RNA and translation in protein, causes the formation of an enzyme in a suitable environment which has the known properties of lysozymes. These enzymes protect the transformed plants against phytopathogenic fungi and animal pests.
• the gene construction to be transferred to the transgenic plants preferably comprises a promoter active in plants, a chimeric gene for T4 lysozyme which contains coding DNA sequences for the signal peptide of barley ⁇ -amylase and the lysozyme of bacteriophage T4, and a polyadenylation signal.
• the amino acid sequence of the T4 lysozyme is taken from phage T4.
• a consensus sequence for the N-linked glycosylation (Asn-X-Ser / Thr) occurring in amino acids 140-142 produces a glycosylated form of the T4 Lysozy protein in plants (Düring, K.; Porsch, P .; Fladung, M .; Lörz, H.; Transgenetic potato plants resistant to the phytopathogenic bacterium Erwina carotovora; The Plant Journal 3, 587-598 (1993)). Due to the glycosylation occurring in planta, which has not been described in patent DE 39 26 390, a change in the enzyme properties or functionality may be caused, which has not been discussed. It is Z. B. possible that the rate of conversion of the enzyme is significantly reduced.
• Patent DE 39 26 390 describes exclusively the use of lysozymes for increasing the resistance of transgenic plants which contain a DNA sequence coding for lysozyme to fungi and animal pests. This takes place with gene constructions which are delimited by the properties described above. These gene constructions do not take into account those possible influencing factors that affect the efficiency of the system in transgenic Plants can diminish. Generation of the T4 lysozyme in transgenic plants as bioreactors for purification and use of T4 lysozyme as a medicament in human and veterinary medicine or as a preservative additive is not considered.
• the aim of the invention is to provide novel proteins and peptides with a wide range of uses and to use them preferably for protection against microbial organisms.
• T4 lysozyme molecule Surprisingly, it has now been found by structure-function analysis of the T4 lysozyme molecule that the antimicrobial activity of the T4 lysozyme is independent of the enzymatic muramidase activity. It was found that T4 lysozyme denatured by heating no longer has enzyme activity but has full antimicrobial activity.
• amphipathic a-helix was identified in the C-terminal part of the T4 lysozyme, which surprisingly alone is sufficient to exert the antimicrobial effect.
• this peptide sequence (amino acids 143-155 in the T4 lysozyme) has no enzyme activity.
• a membrane-interacting function is therefore essential for the antimicrobial effect of lysozymes, e.g. can be exercised through the amphipathic a-helix 143-155.
• proteins and peptides are provided which are derived from lysozyme, in particular from the T4 lysozyme, but no longer contain the lysozyme function ⁇ muramidase activity.
• X denotes any amino acid other than glutamic acid.
• the invention encompasses this protein, parts of this protein and proteins and peptides derived from sequence I by mutation or fragmentation.
• a particularly preferred variant is any mutation of the consensus sequence NQT (amino acids 140 to 142 in the T4 lysozyme). It can thus be achieved that no N-linked glycosylation occurs when expressed in transgenic eukaryotes, which can have a reducing effect on the antimicrobial potential.
• Preferred proteins from sequence I comprise amino acids 12-164 of the T4 lysozyme or parts thereof.
• the invention also relates to short parts of sequence I, preferably peptides, which represent the amino acids 126-141 WDEAAVNLAKSRWYNQ, which can also be mutated in positions 140/141, 143-155 PNRAKRVIFTFRT or at least the amino acids 126-141 WDEAAVNLAKSRWYNQ, which are also in positions 140/141 can be mutated, or contain 143-155 PNRAKRVIFTFRT.
• the invention also extends to recombinant proteins which fuse at least amino acids 126-141 or 143-155 (amphipathic helix) of the T4 lysozyme or partially homologous sequences with the same functionality, which are produced by amino acid exchanges, as a component to other amino acid sequences contain.
• lysozyme obtained from natural sources, preferably the T4 lysozyme in position 11.
• This exchange of the AS glutamic acid present there in the T4 lysozyme with any other AS takes place through conventional protein-technical operations, such as by recloning subfragments, by polymerase chain reaction amplification and modification or by site-directed mutagenesis of certain sections of DNA in the native state or with simultaneous introduction of modifications.
• Lysozyme with proteases the fragments are preferably produced.
• the resulting genes can be used in ' pro- and eukaryotic transgenic organisms for the expression of the proteins or peptides in question.
• the essential biochemical properties resulting from the T4 lysozyme are retained except for the muramidase activity, in particular the bactericidal and fungicidal activity.
• the expression of these recombinant genes can take place in transgenic organisms for the production of the encoded protein or peptide or with the aim of direct antimicrobial activity in vivo.
• sequence I also serves as a protein backbone as an x carrier protein for other antimicrobially active peptides, preferably amphiphatic helices. This ensures that the actually effective elements (short peptides) are stabilized.
• the peptide sequence 143-155 PNRAKRVIFTFRT can be replaced by another such natural or peptide sequence developed by rational design.
• the proteins and peptides produced according to the invention have antimicrobial properties which achieve and in some cases exceed the action of the T4 lysozyme. This is shown below using the example of the survival rates of Escherichia coli cells.
• the table shows that the bactericidal activities of T4 lysozyme and heat-denatured T4 lysozyme do not differ significantly, taking into account that heat-denatured T4 lysozyme does not dissolve 100% even under the selected conditions.
• the heat-denatured T4 lysozyme no longer exhibits enzyme activity (muramidase).
• the mutant M6K in which the 6th amino acid (methionine) is replaced by lysine, already shows a higher bactericidal activity because a hydrophobic amino acid has been replaced by a polar amino acid.
• peptide A4 which comprises amino acids 143-155 of the T4 lysozyme (in sequence I)
• has no enzymatic murimidase activity but has a significantly higher bactericidal activity.
• these example substances have a fungicidal activity against germinating zoospores of Phytophthora nicotianae.
• Peptide A23 comprises AA 126-141 of T4 lysozyme buffer I: 20 ⁇ l buffer AI + 1 ⁇ l PBS buffer II: 20 ⁇ l buffer AI + 1 ⁇ l 50% DMSO / 50% PBS buffer III: 20 ⁇ l buffer AI + 1 ⁇ l 40% DMSO / 50% PBS, 0.3 ; Triton X-100
• proteins and peptides according to the invention or the unprocessed material of a transgenic organism can be used, for example, as an additive for food and feed or for other substances in order to prevent the growth of To prevent microorganisms.
• these proteins and peptides can also be used in medical therapies in, for example, infectious diseases.
• Another area of application is cancer therapy, since cancer cells have a similar cell membrane structure to bacterial cells, in contrast to membranes of healthy eukaryotic cells, which differ significantly in structure.
• the fields of application for this invention are very large due to the ubiquitous occurrence of diseases caused by microorganisms. They range from human and veterinary medicine to resistance breeding in plants and the preventive use of bactericidal and fungicidal additives in food.
• the properties of the proteins and peptides covered by the invention also enable further fields of application which are not associated with infections by microorganisms, e.g. cancer therapy.
• the use of smaller partial sequences which are preferably derived from the C-terminal part of the T4 lysozyme, can have particular advantages, for example better tissue penetration, lower allergenicity potential due to smaller size, higher antimicrobial activity, etc. to have.
• a mixture of peptide fragments is generated by digestion with suitable proteases (e.g. clostripain, pepsin, trypsin), which is then separated into the individual fragments using chromatographic methods (reversed phase HPLC with C18 column).
• proteases e.g. clostripain, pepsin, trypsin
• the bactericidal activity of individual fragments is determined by incubation (use of 1-10 ⁇ g protein or peptide for a quantity of lxl ⁇ bacterial cells) for one hour with Escherichia coli or other gram-negative (e.g.
• fungicidal activity of individual fragments is determined by incubation for 20 hours with spores of Phytophthora nicotianae or other types of fungus and subsequent plating out to determine the reduced growth in length of the fungus hyphae.
• the relative activity to purified T4 lysozyme is determined as the standard.
• Protein or peptide partial sequences from the T4 lysozyme can also be synthesized chemically, for example the amphipathic ⁇ -helix 143-155.
• the bactericidal and fungicidal effects are determined as described above. 3. Production of the peptides or proteins by genetic engineering
• the coding DNA sequences for the C-terminal half of the T4 lysozyme from amino acid 74 to amino acid 164 are cloned as a subfragment or for the amphipathic helix of amino acids 143 to 155 are isolated and cloned by PCR amplification.
• Site-directed mutagenesis allows the consensus sequence for N-glycosylation to be mutated in such a way that N-linked glycosylation no longer takes place / e.g. Thr 142 ⁇ Ala 142).
• the recombinant genes generated are cloned under the control of suitable promoters, so that expression in the transgenic pro- or eukaryotic organism provided is possible. This process will be explained using two examples.
• the recombinant gene produced is cloned into a bacterial expression vector (for example from the pQE series, Qiagen).
• a so-called tag-peptide sequence for example a 6xHIS tag or a c- myc tag or a strep tag.
• a controlled biosynthesis of the proteins or peptides according to the invention can take place under controlled conditions under the control of an inducible promoter (eg Tac promoter).
• the protein or peptide according to the invention is isolated, purified with the aid of the 6x HIS tag simply by means of affinity chromatography using nickel chelate columns, after which a highly pure product is obtained.
• transgenic dicotyledonous plants is chosen for expression in transgenic eukaryotes.
• the recombinant genes which code for the proteins or peptides according to the invention are cloned under the control of promoters which are constitutively or regulatably active in plants (for example cauliflower mosaic virus 35S promoter, Agrobacterium tumefaciens mannopin synthase promoter, maize GapC4 promoter, potato ubiquitin promoter).
• a terminator sequence for example cauliflower mosaic virus 35S terminator, Agrobacterium tumefaciens nopalin synthase terminator, Agrobacterium tumefaciens octopine synthase terminator
• This expression cassette is transferred into a binary vector (eg pBIN 19, pPCV701, pSR 8-30, pSR 8-35 / 1) which is suitable for gene transfer by means of Agrobacterium tumefaciens.
• Plant explants with these genetically modified agrobacteria are used for gene transfer into the plant.
• the transformation of plants can also be carried out with all other suitable methods (e.g. with the particle cannon).
• the incorporation of the foreign genes in transgenic plants can be done by suitable restriction digestion of the isolated genomic DNA and subsequent Southern hybridization or by amplification of the foreign DNA sequence using the polymerase chain reaction.
• the transcription of the genes in mRNA can be detected in the Northern blot or by other suitable methods.
• the translation of the genes into the encoded proteins can be examined and detected by Western blot or by differently structured ELISA tests or by other suitable methods.
• the presence of the protein or peptide according to the invention can thereby be proven. Its biological activity is determined by the methods described above.
• its biological effectiveness can also be determined by resistance tests on transgenic plants.
• the proteins and peptides according to the invention can also be overexpressed for production in transgenic plants.

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• 1997
  • 1997-11-05 DE DE19749973A patent/DE19749973C1/de not_active Expired - Fee Related
• 1998
  • 1998-10-31 NZ NZ504368A patent/NZ504368A/xx unknown
  • 1998-10-31 WO PCT/DE1998/003287 patent/WO1999024589A2/de not_active Application Discontinuation
  • 1998-10-31 JP JP2000520583A patent/JP2002503443A/ja active Pending
  • 1998-10-31 CA CA002308618A patent/CA2308618A1/en not_active Abandoned
  • 1998-10-31 EP EP98963336A patent/EP1029061A2/de not_active Withdrawn
  • 1998-10-31 AU AU18679/99A patent/AU751422B2/en not_active Ceased
  • 1998-10-31 IL IL13598798A patent/IL135987A0/xx unknown
• 2000
  • 2000-05-05 US US09/567,563 patent/US6515106B1/en not_active Expired - Fee Related

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