Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-05-05
2003-02-04
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S206000, C435S440000, C536S023200, C536S023100
Reexamination Certificate
active
06515106
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polypeptides exhibiting antibiotic, and anti-viral and anti-cancer effects. In particular, the invention relates to modified T4 lysozyme, polypeptides comprising fragments of T4 lysozyme and to the production and use thereof. The fields of application for this invention are wide-ranging, and include, for example, human and veterinary uses, resistance cultivation in plants and prevention of bacterial and/or fungal-mediated food spoilage.
BACKGROUND OF THE INVENTION
T4 lysozyme is a protein which is formed from bacteriophage T4. T4 lysozyme functions to open the bacterial host cell after it has been propagated therein, and to allow it to enter the environment. It has been assumed that the enyzme activity (muramidase) of T4 lysozyme destroys the bacteria (Tsugita, A. (1971) Phage lysozyme and other lytic enzymes; in: Boyer, P. D. (ed.) The Enzymes, Vol. V. Academic Press, New York, p. 344-411). T4 lysozyme is known to effect a specific cleavage between C-1 of the muramic acid group and C-4 of acetylglucosamine. This cleavage ruptures the bacterial muramine network ruptures and destabilizes the cell wall. However, the lysozyme must first traverse the bacterial cell membrane to access its substrate, the muramine layer. Until now it has not been possible to explain the transport route of the T4 lysozyme through the inner cell membrane to the muramine layer. It has been assumed that, as a consequence of rupture of the muramine layer the bacteria burst open through destabilization and are thus destroyed.
Further enzymatic or other biochemical functions, e.g. membrane affinities, of the T4 lysozyme have not previously been described.
DE 39 26 390 describes the introduction of lysozyme genes as exogenic or additional DNA into plants, to increase resistance to fungi and animal microbes.
Lysozyme genes are understood here to be any nucleic acids (e.g., DNA) that encode lysozymes. Lysozymes protect the transformed plants against plant-pathogenic fungi and animal microbes.
The nucleic acid used to transform plants preferably includes a promotor active in plants, a chimeric gene for T4 lysozyme, containing the coding DNA sequences for the signal peptide of alpha-amylase from barley and for the lysozyme of the bacteriophage T4, and a polyadenylation signal. The amino acid sequence of the T4 lysozyme is obtained from the phage T4.
Through a consensus sequence for the N-bound glycosylation (Asn-X-Ser/Thr) existing in the amino acids 140-142, a glycosylated form of the T4 lysozyme protein is produced in plants (Düring, K.; Porsch, P.; Fladung, M.; Lörz, H.; Transgenetic potato plants resistant to the phytopathogenic bacteria
Erwina carotovora;
The Plant Journal 3, 587-598 (1993) ). The glycosylation occurring in planta, which has not been described in patent DE 39 26 390, can be the cause of a change in the enzyme properties or functionality. It is possible, for example, that the conversion rate of the enzyme is significantly reduced.
DE 39 26 390 describes only the utilization of lysozymes for increasing the resistance of transgenic plants, containing a lysozyme-coding DNA sequence, to fungi and animal microbes. The described gene constructions are limited by the aforementioned problems. Moreover, these gene constructions do not solve the problem of potential influencing factors, which can reduce the efficiency of the described system in transgenic plants. Production of the T4 lysozyme in transgenic plants, and use of T4 lysozyme as a medical remedy in human or veterinary medicine, or as a preservative additive is not considered.
The mode of action of the T4 lysozyme (for which at the time of registration of patent DE 39 26 390 only the muramidase activity was known) on fungi and other microbes has still not been explained. The definition of lysozyme genes given in patent DE 39 26 390 relates only to translation into proteins which possess the known properties of lysozymes.
There is a need in the art for new types of polypeptides with a wide range of useful properties, such as antibiotic, anti-viral and anti-cancer properties.
SUMMARY OF THE INVENTION
The invention generally relates to antibiotic polypeptides comprising a modified T4 lysozyme, or a functional equivalent thereof, which exhibits antibiotic activity without exhibiting muramidase activity. The polypeptide preferably does not comprise full-length, native T4 lysozyme.
In a preferred aspect, the native glutamic acid residue at position 11 of the T4 lysozyme is replaced by any amino acid residue other than glutamic acid.
In another preferred aspect, the segment is selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, and fragments and derivatives thereof, wherein X is any amino acid residue except glutamic acid.
A preferred set of segments includes:
amino acids 12-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 126-141 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 143-155 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 74-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 114-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof; and
amino acids 124-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof.
Another preferred group of functional equivalents includes:
amino acids 12-164 of SEQ ID NO: 1 comprising one or more mutations at any one or more of positions 140-142, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
amino acids 126-141 of SEQ ID NO: 1 comprising one or more mutations at any one or more of position s 140-141, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
amino acids 74-164 of SEQ ID NO: 1 comprising one or more mutations at any one or more of positions 140-142, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
amino acids 114-164 of SEQ ID NO: 1 comprising one or more mutations at any one or more of positions 140-142, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
The antibiotic polypeptide may also consist of the above-listed sets of segments, subsegments and functional equivalents.
The invention also relates methods for producing the segments, subsegments and functional equivalents (e.g., proteolysis of the native protein, chemical synthesis, recombinant production).
In another aspect, the invention relates to a nucleic acid encoding a polypeptide comprising a segment of T4 lysozyme, or a functional equivalent of said segment, which polypeptide exhibits antibiotic activity but which does not exhibit muramidase activity. The recombinant nucleic acid preferably does not encode full-length T4 lysozyme.
The invention also relates to a method for killing a microbe and/or controlling a population of microbes. The method generally comprises bringing the microbe and/or population of microbes into contact with a polypeptide comprising a segment of T4 lysozyme, or comprising a functional equivalent of said segment, which polypeptide exhibits antibiotic activity but which does not exhibit muramidase activity. In one aspect, the method is used to kill a bacteria or fungus, or to eliminate or control a bacterial colony or a fungal colony. In another aspect, the microbe is a pathogen of an organism and the organism is genetically modified to produce the polypeptide, thereby bringing the pathogenic organism and/or colony into contact with the polypeptide, to effect its antibiotic effect on the organism or colony. In a preferred aspect, the microbe is a plant pathogen and the polypeptide is applied to the plant and/or the plant is genetically modified to produce the polypeptide.
The invention provides microbe control compositions comprising antibiotic polypeptides formulated for application to the target microbes or their situs.
Antibiotic compositions of the invention generally contain one or more of the antibiotic polypeptides described above and a carrie
Achutamurthy Ponnathapu
Fuierer Marianne
Hultquist Steven J.
MPB Cologne GmbH Molecular Plant and Protein Biotechnology
Pak Yong
LandOfFree
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