JP2011072294A - New antibacterial peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a thanatin derivative having an antibacterial activity superior to that of the wild type thanatin and being producible at low cost. <P>SOLUTION: The antibacterial peptide, consisting of only natural amino acids, is producible at low cost by the gene recombination technology. Furthermore, the peptide shows the antibacterial activity higher than that of the wild type thanatin, so the peptide is widely used as an antibacterial agent. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel antibacterial peptide and use thereof, and more particularly to a tanatine substituted peptide having antibacterial activity and a method for producing the same.

  Antibacterial peptides are peptides consisting of 10 to several tens of amino acid residues, and are attracting attention as molecules that function in the innate immune system of living organisms. Many antibacterial peptides produced by microorganisms such as bacteria, plants, insects, amphibians, mammals and the like have been reported so far (Patent Documents 1 to 4, etc.). Antibacterial peptides are expected to be used as antibacterial agents because they have a wide range of antibacterial activity compared to antibiotics and are resistant to the generation of resistant bacteria.

  Tanatin is an antibacterial peptide consisting of 21 amino acid residues isolated from stink bugs, and is known to have a relatively strong bactericidal activity among antibacterial peptides and a wide range of antibacterial activities against gram-positive bacteria, gram-negative bacteria, and fungi. (Non-Patent Document 1). Because of these characteristics, although tanatine is expected to be used as an antibacterial agent, the antibacterial activity of wild-type tanatine is insufficient in practice, and it is necessary to enhance the antibacterial activity.

  Studies on tanatine derivatives with enhanced antibacterial activity of tanatine are underway. Non-Patent Document 2 and Non-Patent Document 3 describe chemically modified tanatine in which a methyl group, an ethyl group, a ter-butyl group, and an octyl group are introduced into the cysteine residues present at the 11th and 18th positions on the N-terminal side of tanatine. Is reported to have higher antibacterial activity against the Gram-positive micrococcus luteus than wild-type tanatine.

  Although chemically modified tanatine as described above has high antibacterial activity, it has an antibacterial peptide that is composed of natural amino acids and has high safety to the human body because it has an artificial functional group introduced into the peptide. One may be lost. Furthermore, since the production of chemically modified tanatine involves a chemical synthesis process, there is a problem that the production cost increases.

  On the other hand, tanatin derivatives composed only of natural amino acids by substitution of amino acid residues have also been reported. In Non-Patent Document 4, a tanatin derivative in which 1 to 3 amino acid residues from the 2nd position to the 21st position are substituted with natural amino acids, and in Patent Document 5, in the wild type, an intramolecular S—S bond is formed. In the non-patent document 2, a tanatin derivative in which the cysteine residues at the 11th position and the 18th position are substituted with serine in the non-patent document 2, Although each is disclosed, none of the antibacterial activities superior to wild-type tanatine has been recognized.

Japanese Patent Laid-Open No. 2-270897 JP 2000-245483 A Japanese translation of PCT publication No. 2002-522556 JP 2007-274918 A Japanese Patent Laying-Open No. 2005-281591

Fehlbaum et al. , Proc. Natl. Acad. Sci. , 93, 1221-5 (1996) Imamura et al. Biochem. Biophys. Res. Commun. , 369, 609-15 (2008) Orikasa et al. Biosci. Biotechnol. Biochem. , 73, 90183-1-2 (2009) Taguchi et al. , J .; Biochem. , 128, 745-754 (2000)

  An object of the present invention is to provide a tanatine derivative that has antibacterial activity superior to that of wild-type tanatine and can be produced at low cost.

  The present inventors have found that modified tanatine in which one or both of cysteine residues at the 11th and 18th positions of N-terminal side of tanacin is substituted with phenylalanine, leucine, or isoleucine exhibits high antibacterial activity. Each invention was completed.

(1) In the polypeptide represented by the amino acid sequence shown in SEQ ID NO: 1, one or both of the cysteine residues at the 11th and 18th positions on the N-terminal side are substituted with any of phenylalanine, leucine, and isoleucine Antibacterial peptide.

(2) The antibacterial peptide according to (1), wherein one or more amino acid residues on the N-terminal side or C-terminal side are deleted.

(3) A vector comprising a polynucleotide encoding the polypeptide according to (1) or (2).

(4) A transformant comprising the vector according to (3).

(5) The transformant according to (4), wherein the transformant is a plant.

(6) A method for producing an antibacterial peptide, wherein the vector according to (3) or the transformant according to (4) or (5) is used.

(7) An antibacterial agent comprising the antibacterial peptide according to (1) or (2).

  Since the antibacterial peptide of the present invention is composed only of natural amino acids, it can be produced at low cost using genetic recombination technology, and further exhibits high antibacterial activity compared to wild-type tanatine. It can be used as an agent. In addition, the antibacterial peptide of the present invention has a reduced antibacterial action against gram-negative bacteria, and is advantageous when recombinantly producing gram-negative bacteria as a host.

  The antibacterial peptide of the present invention has an amino acid sequence in which one or both of the cysteine residues at the 11th and 18th positions on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 are substituted with any of phenylalanine, leucine and isoleucine. It is polypeptide which has. Either or both of the cysteine residues at positions 11 and 18 can be substituted. When both are substituted, phenylalanine, leucine, and isoleucine may be substituted with the same amino acid. You may substitute by an amino acid. A preferred polypeptide is a polypeptide having an amino acid sequence in which both the 11th and 18th cysteine residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 are substituted with either phenylalanine, leucine or isoleucine. is there.

  The antibacterial peptide of the present invention has an amino acid sequence in which one or both of the amino acids at positions 11 and 18 are substituted with any of phenylalanine, leucine, and isoleucine in addition to the above polypeptide, and maintains antibacterial activity. As long as the amino acid residue on the N-terminal side or C-terminal side is deleted, one or more amino acid residues may be deleted. The deletion is preferably a deletion of the first to ninth amino acid residues on the N-terminal side, and a deletion of the first to third amino acid residues counted from the C-terminal side on the C-terminal side. More preferably, the deletion is a deletion of the first to seventh amino acid residues.

  The polynucleotide encoding the antimicrobial peptide of the present invention is preferably DNA. By incorporating this into an appropriate expression vector, the vector of the present invention can be obtained. As an expression vector, an appropriate vector may be selected and used according to the host and purpose to be used. In addition to various plasmids such as pUC, pET, and pBi, bacteriophage, baculovirus, retrovirus, vaccinia It is also possible to use various viruses such as viruses.

  In addition to the polypeptide encoding the antimicrobial peptide of the present invention, the expression vector encodes an enhancer sequence, a promoter sequence, a ribosome binding sequence, a base sequence used for the purpose of amplification of copy number, and a signal peptide, if necessary. Other base sequences such as a base sequence encoding the other polypeptide, a base sequence encoding another polypeptide, a poly A addition sequence, a splicing sequence, a replication start point, and a base sequence of a gene serving as a selection marker.

  Examples of promoter sequences include T7 promoter, lac promoter, trp promoter, λPL promoter, etc. when the host is E. coli, and PHO5 promoter, GAP promoter, ADH promoter, etc. when the host is yeast. In the case of insect cells, polyhedrin polomotor, p10 promoter, etc., and when the host is an animal cell, SV40-derived promoter, retrovirus promoter, cytomegalovirus (human CMV) IE (immediate early) gene When the host is a plant cell, cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase gene promoter (Pnos), tobacco-derived PR protein promoter, etc. It can gel.

  For gene recombination, a translation initiation codon and translation termination codon are added to the polynucleotide encoding the antibacterial peptide of the present invention using an appropriate synthetic DNA adapter, or a suitable restriction enzyme cleavage sequence is newly generated in the base sequence. Or it can be eliminated. These are within the scope of operations usually performed by those skilled in the art, and those skilled in the art can arbitrarily and easily process them based on the polynucleotide encoding the antimicrobial peptide of the present invention.

  The transformant of the present invention can be obtained by introducing the expression vector into an appropriate host cell. In the present specification, the “transformant” includes cells, tissues, organs and individual organisms. The host cell is not particularly limited as long as the antibacterial peptide of the present invention is expressed, and microorganisms such as bacteria and yeast, insect cells, mammalian cells, plant cells, and the like can be used.

  The transformation method for introducing the expression vector into the host cell is not particularly limited, and known methods such as electroporation method, protoplast method, alkali metal method, calcium phosphate precipitation method, DEAE dextran method, microinjection method, particle gun method The Agrobacterium method can be used. The polynucleotide introduced by transformation may be integrated into the host genomic DNA, or may be present in an expression vector.

  Surprisingly, it has been confirmed that the antibacterial peptide of the present invention reduces the antibacterial activity of gram-negative bacteria possessed by wild-type tanatine and increases the specificity of the antibacterial spectrum. Since the Agrobacterium is a Gram-negative bacterium, when the plant is transformed using the Agrobacterium as a vector to produce a transformed plant imparted with antibacterial activity, the peptide of the present invention is used as a host. This is advantageous in that it does not hinder the growth of the rice.

  The transformant of the present invention can be used as a plant having improved disease resistance when an organism imparted with antibacterial properties, such as a plant, is used as a host. Furthermore, since the transformant of the present invention expresses the antibacterial peptide, the antibacterial peptide of the present invention can be obtained by culturing, cultivating or breeding and then collecting and purifying from the culture. As a recovery / purification method, an appropriate method can be appropriately selected from known methods. That is, an appropriate method is appropriately selected from commonly used methods such as filtration, centrifugation, cell disruption, gel filtration method, ion exchange chromatography and the like, and if necessary, purified in an appropriate order using an HPLC system or the like. Just do it.

  In addition, when the antibacterial peptide of the present invention is expressed as a fusion protein with another functional protein or polypeptide, it is preferable to employ a purification method characteristic of the functional protein or polypeptide. The fusion protein can be cleaved with an appropriate protease (thrombin, trypsin, etc.) to recover the antimicrobial peptide of the present invention. In addition, a method obtained by a cell-free synthesis method using a recombinant DNA molecule is one of the methods for producing by genetic engineering.

  In addition to the genetic engineering method as described above, the antimicrobial peptide of the present invention can be produced by a known peptide production method such as peptide synthesis. Examples of the production method by peptide synthesis include organic chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method), and suitable peptide synthesis available on the market. It can also be manufactured using a machine.

  The antibacterial activity of the antibacterial peptide of the present invention can be measured using a growth inhibition test using microorganisms, for example, the test method described in Non-Patent Document 1. As the microorganism, known gram-positive bacteria, gram-negative bacteria, fungi and other microorganisms having tanatin sensitivity can be used, but the gram-positive bacteria Micrococcus luteus or Corynebacterium glutamicum and the gram-negative bacteria Escherichia coli or Acinetobacter baumanni are used. Is preferred.

  As the antibacterial agent of the present invention, the antibacterial peptide may be used as it is, or the antibacterial peptide may be used in a form contained in the above-described transformant, culture thereof or the like. In addition, these can be combined with general excipients to form a composition, and further formulated into a general external dosage form, internal preparation, injection or other general dosage form. Excipients used in the formulation include, for example, oral solid preparations such as tablets and capsules, internal liquid preparations such as aqueous solutions and suspensions, ointments, patches, lotions, creams, sprays, suppositories, and other agents. Components that are widely known and used by those skilled in the art for each mold can be used in appropriate combinations.

  In addition to antibacterial peptides, the above-mentioned composition or various dosage forms may contain other antibiotics and other ingredients as necessary, and can be used as pharmaceuticals, quasi-drugs, and agricultural chemicals, and can be safely added to food. You may add to food and drink as a thing. Furthermore, the antibacterial agent of the present invention can be used for the manufacture of articles such as daily necessities imparted with antibacterial properties by coating, spreading, fixing, etc. on the surface of the article.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail, this invention is not limitedly limited to this Example.

<Example>
1) All four types of polypeptides (wild type: SEQ ID NO: 1, mutant L: SEQ ID NO: 2, mutant I: SEQ ID NO: 3, mutant F: SEQ ID NO: 4) having the amino acid sequence shown in Table 1 Solid phase synthesis (synthesized using a peptide synthesizer.

2) According to the MIC test method described in Non-Patent Document 2 (Imamura et al.), The minimum concentration of four types of polypeptides synthesized using Micrococcus luteus, Corynebacterium glutamicum, Escherichia coli, and Acinetobacter baumannii were measured. did. The results are shown in Table 2.

  Gram-positive bacteria For luteus, all three variants are twice the wild type, C.I. For glutamicum, variant F showed twice the antimicrobial activity of the wild type. In addition, the activity of Gram-negative bacteria decreased or disappeared, indicating that the specificity of the antibacterial spectrum was increased.

Claims (7)

In the polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 1, an antimicrobial peptide in which one or both of the cysteine residues at the 11th and 18th positions on the N-terminal side are substituted with any of phenylalanine, leucine, and isoleucine. The antibacterial peptide according to claim 1, wherein one or more amino acid residues at the N-terminal side or C-terminal side are deleted. A vector comprising a polynucleotide encoding the polypeptide according to claim 1 or 2. A transformant comprising the vector according to claim 3. The transformant according to claim 4, wherein the transformant is a plant. A method for producing an antibacterial peptide, wherein the vector according to claim 3 or the transformant according to claim 4 or 5 is used. An antibacterial agent comprising the antibacterial peptide according to claim 1 or 2.