Definitions

• the present invention relates to a method for mass production of an antimicrobial peptide, more specifically, to a method for mass production of an antimicrobial peptide by producing the antimicrobial peptide in a form of fusion protein with a foreign peptide through gene manipulation.
• antimicrobial peptides do not easily lost their biological activities by physical and chemical factors such as heat, alkali, etc. Moreover, they do not readily induce a resistance to microorganisms as they show an antimicrobial activity through their characteristic action mechanism which is clearly discriminated from conventional antibiotics. Thus, antimicrobial peptides have enjoyed high industrial applicability in the areas of pharmacy, food, etc. However, there is a crucial problem in the industrial application of the antimicrobial peptides, since the conventional techniques do not permit mass production of the peptides in a low price.
• USP 5,593,866 teaches a process for preparing a positively charged antimicrobial peptide as a fusion protein with a negatively charged peptide to inhibit bacterial proteolysis, where glutathione-S-transferase, protein A, IgG-binding domain of protein A, protein F from Pseudomonas aeru ⁇ inosa or prepro defensin is employed as the negatively charged peptide.
• the present inventors have made an effort to solve the disadvantages of low productivity and poor economy in the course of manufacturing the antimicrobial peptide, and successfully prepared the antimicrobial peptide in a massive and economical manner by the aid of recombinant DNA technology.
• a primary object of the present invention is to provide a method for mass production of an antimicrobial peptide in recombinant microorganisms, which employs an expression system permitting mass production of the antimicrobial peptide.
• Figure 1(A) shows a nucleotide sequence(SEQ ID N0:1) of Guamerin gene and amino acid sequence translated therefrom (SEQ ID NO: 2) .
• Figure 1(B) shows a nucleotide sequence (SEQ ID NO: 3) of MMIS (modified magainin intervening segment) gene and amino acid sequence translated therefrom(SEQ ID N0:4).
• Figure 2 (A) is a schematic diagram showing a construction strategy of a fusion product (SEQ ID NO: 5) of a Guamerin gene with a Buforin II gene by PCR.
• Figure 2 (B) is a schematic diagram showing a construction strategy of fusion product (SEQ ID NO: 6) of a MMIS gene with a Buforin II gene by PCR.
• Figure 3 is a schematic diagram showing a construction strategy of a multimeric fusion gene using a gene amplification vector.
• Figure 4 (A) is a schematic diagram showing a construction strategy of a fusion gene (gene I) (SEQ ID NO: 7) containing a gene of an antimicrobial peptide MSI-78 and a gene of Guamerin.
• Figure 4 (B) is a schematic diagram showing a construction strategy of a fusion gene (gene II) (SEQ ID NO: 8) containing a gene of an antimicrobial peptide MSI-78 and a gene of Guamerin.
• Figure 5(A) is SDS-PAGE pattern of cell lysates of E. coli transformed with vectors containing multimeric fusion gene comprising
• Figure 5(B) is SDS-PAGE pattern of cell lysates of E. coli transformed with vectors containing prepromagainin gene after induction of protein expression.
• Figure 6 is SDS-PAGE pattern of cell lysates of E. coli transformed with vectors comprising fusion genes containing Guamerin gene and genes of various antimicrobial peptides, after induction of protein expression.
• Figure 7 is SDS-PAGE pattern of cell lysates of E. coli transformed with vectors comprising fusion genes containing Guamerin gene and genes of various antimicrobial peptides, after induction of protein expression.
• a method for mass production of an antimicrobial peptide of the present invention comprises the steps of: constructing a fusion gene containing a first gene encoding an acidic peptide having at least two cysteine residues and a second gene encoding a basic antimicrobial peptide; transforming a host microorganism with an expression vector comprising the fusion gene; culturing the transformed microorganism to express a fusion peptide containing the acid peptide and the antimicrobial peptide; and, recovering the antimicrobial peptide from the fusion peptide.
• a gene construct which comprises a first gene encoding an acidic peptide having at least two cysteine residues and a second gene encoding a basic antimicrobial peptide, and an expression vector which comprises a promoter operably linked to a gene sequence containing a first gene encoding an acidic peptide having at least two cysteine residues and a second gene encoding a basic antimicrobial peptide, are essentially required, and the fusion gene may be present in a form of monomer or multimer.
• a first gene codes for an acidic peptide having at least two cysteine residues and neutralizing positive charges of an antimicrobial peptide substantially.
• the length of the acidic peptide is not limited, it is, preferably, equal to or longer than that of the antimicrobial peptide in order to efficiently neutralize the charges of a desired antimicrobial peptide, when considering length and distribution of positive charges.
• the acidic peptide has two or more cysteine residues. It is postulated that the cysteine residues promote interaction between negative charges of the acidic peptide and positive charges of the antimicrobial peptide by the formation of a secondary structure through disulfide bonds.
• the acidic peptide may be synthesized artificially or selected among natural acidic peptides, and may be obtained using the synthetic gene encoding the peptide or isolated from nature.
• the artificially designed acidic peptide has two or more cysteine residues, and the natural acidic peptide may be modified to have sufficient cysteine residues.
• the acidic peptide gene may be modified in various ways for the purpose of easy fusion with a second gene encoding the antimicrobial peptide, easy isolation of the antimicrobial peptide from the fusion peptide, or the preparation of various multimeric forms of a fusion gene.
• the acidic peptide gene may be synthesized or modified so that it can be connected to the antimicrobial peptide gene to have a correct reading frame resulting the desired antimicrobial peptide.
• the acidic peptide gene may be synthesized or modified to include nucleotide sequences encoding a cleavage site for a specific protease or a chemical in order to isolate the antimicrobial peptide from the expressed fusion peptide.
• the acidic peptide gene may be selected to have the most suitable length for the neutralization of the antimicrobial peptide among monomers or multimers of the acidic peptide genes.
• the multimer of an acidic peptide gene may be prepared by employing gene amplification technique.
• vectors comprising multimers of an acidic peptide gene can be prepared by inserting an acidic peptide gene between two Class-IIS restriction enzyme sites of a vector containing two oppositely oriented Class-IIS restriction enzyme sites, digesting the vector with a Class-IIS restriction enzyme, isolating a DNA fragment containing the acidic peptide gene, self-ligating the isolated DNA fragments to prepare multimers, and cloning the various multimers into the vector digested with the Class-IIS restriction enzyme (see: Lee, J.H. et al . , Genetic Analysis: Biomolecular Engineering, 13:139-145(1996)).
• the antimicrobial peptide may be designed artificially or selected among natural acidic peptides, and may be obtained using the synthetic gene encoding the desired peptide or isolated from nature.
• the antimicrobial peptide gene may be modified in various ways for the purpose of easy fusion with the acidic peptide gene, easy isolation of the antimicrobial peptide from the fusion peptide, or the preparation of various multimeric forms of the fusion gene.
• the antimicrobial peptide gene may be modified so that the C-terminal region of the antimicrobial peptide can be connected to the N-terminal region of the acidic peptide in a correct reading frame (antimicrobial peptide gene I) .
• the antimicrobial peptide gene may be modified to include nucleotide sequences encoding a site cleaved by a specific protease or a chemical at the N-terminus in order to isolate the antimicrobial peptide from the expressed fusion peptide, and nucleotide sequences permitting termination of peptide synthesis at the C- terminus of the antimicrobial peptide (antimicrobial peptide gene II) .
• the antimicrobial peptide gene may be modified to include nucleotide sequences encoding a cleavage site for a specific protease or a chemical (for example, a codon encoding a methionine residue for the cleavage by CNBr) at the N-terminus and the C-terminus of the antimicrobial peptide in order to isolate the antimicrobial peptide from the expressed fusion peptide (antimicrobial peptide gene III) .
• the fusion gene may be prepared by ligating the acidic peptide gene and the antimicrobial peptide gene prepared as described above, and the acidic peptide gene or the antimicrobial peptide gene may be a monomer or a multimer as mentioned above.
• the fusion gene contains a first gene encoding an acidic peptide and a second gene encoding an antimicrobial peptide gene, and those may be ligated directly or indirectly through linker, etc., if the two genes are connected in a correct reading frame .
• the fusion gene may be prepared by modifying the acidic peptide gene and the antimicrobial peptide gene to have complementary nucleotide sequences at the 3 '-termini of single strands of each gene, annealing two genes via a partial hybridization, and performing PCR with the hybridized genes as a template and with primers corresponding to the sequences to the 5'- termini of respective single stranded genes.
• Various number of monomer of a fusion gene thus prepared may be concatenated to prepare various multimers of the fusion gene by the conventional methods in the art, e.g., self-ligation of a fusion gene.
• a multimer of a fusion gene may be also prepared by employing gene amplification system.
• vectors containing multimers of a fusion gene can be prepared by inserting the fusion gene between two Class-IIS restriction enzyme sites of a vector containing two oppositely oriented Class-IIS restriction enzyme sites, digesting the vector with the Class-IIS restriction enzyme, isolating a DNA fragment containing the fusion gene, self-ligating the isolated DNA fragments to prepare multimers of a fusion gene, and cloning the multimers of the fusion gene into the vector digested with the Class-IIS restriction enzyme.
• a multimer of a fusion gene is a multimer of the fusion gene comprising antimicrobial peptide gene III.
• a multimer of a fusion gene is a multimer wherein the fusion gene comprising antimicrobial peptide gene II is ligated to 3'- terminus of a monomer or multimer of a fusion gene comprising antimicrobial peptide gene I.
• a multimer of a fusion gene is a multimer wherein the fusion gene comprising antimicrobial peptide gene II is ligated to 3'- terminus of a monomer or multimer of a fusion gene comprising antimicrobial peptide gene III.
• a multimer of a fusion gene can be cloned into a suitable expression vector and expressed in a microorganism, e.g., E. coli, to express multimer of the fusion peptide.
• the multimer of the fusion peptide is treated with an enzyme or a chemical, e.g., CNBr, to remove the acidic peptide and separate the antimicrobial peptide into monomers, and the antimicrobial peptide is purified using cation-exchange chromatography, etc.
• the antimicrobial peptide present at the end of the multimer may be obtained in a monomer of native form.
• the present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention. Particularly, since antimicrobial peptides, acidic peptides and genes of their multimers used in Examples are only preferred embodiments of the invention, the present invention covers all of the inventions employing an acidic peptide containing at least two cysteins residues for the purpose of mass production of various basic antimicrobial peptides.
• G The native Guamerin
• M a modified MIS
• oligonucleotides thus synthesized were dissolved in TE buffer (pH 8.0) in the same molar ratio, heated at 70°C for 10 minutes, and left to stand at 0°C for 30 minutes. After 20%(w/v) polyacryla ide gel electrophoresis, double stranded DNA fragments were isolated, and cloned into pBBSl vector (see: Lee, J.H. et al . , Genetic Analysis: Biomolecular Engineering, 13:139-145(1996)) digested with Bbsl to construct pBBSl-Gi (Guamerin) or pBBSl-Mi (MIS) vector.
• pBBSl vector see: Lee, J.H. et al . , Genetic Analysis: Biomolecular Engineering, 13:139-145(1996)
• methionine codons were introduced to both ends of a gene of Buforin II (TRSSRAGLQFPVGRVHRLLRK(SEQ ID NO:9); Park, CB. et al . , (1996) Biochem. Biophys. Res. Co m., 218, 408-413), an antimicrobial peptide.
• a DNA sequence encoding Buforin II (hereinafter, referred to as "B") was synthesized and cloned into pBBSl vector digested with Bbsl to construct pBBSl-Bl vector.
• the resulting pBBSl-Bl vector contains a complete Buforin II gene and two methionine codons at both ends of the Buforin II gene.
• Example 3 Preparation of a fusion gene containing an acidic peptide gene and an antimicrobial peptide gene
• PCR was carried out as followings (see: Figures 2 (A) and 2(B)): Using a couple of primers corresponding to 5 '-end and 3 '-end of the acidic peptide (i. e., Guamerin) gene (primer 1: 5'- AAAGAAGACGGCCCCCGGTCGACGAGAATGCG-3' (SEQ ID NO: 10) and primer 2: 5' -GCTGCTACGGGTCATGATCCCCGCGCAGGT-3 ' (SEQ ID NO:ll)), respectively, the Guamerin gene was amplified by the aid of PCR technique.
• primers corresponding to 5 '-end and 3 '-end of the acidic peptide i. e., Guamerin
• primer 1 5'- AAAGAAGACGGCCCCCGGTCGACGAGAATGCG-3' (SEQ ID NO: 10
• primer 2 5' -GCTGCTACGGGTCATGATCCCCGCGCAGGT-3 ' (SEQ ID NO:ll)
• the PCR products thus amplified were mixed in a same molar ratio and amplified again by PCR using primer 1(SEQ ID NO: 10) and primer 4 (SEQ ID NO: 13).
• the PCR product thus obtained was digested with Bbsl.
• the fragments of the fusion gene containing the Guamerin gene and the Buforin II gene were isolated and cloned into pBBSl vector digested with Bbsl to construct pBBSl-(GB)l vector (see: Figure 2 (A) ) .
• the pBBSl- (GB) 1 vector was digested with Bbsl and the fragments containing the fusion gene were isolated.
• MIS containing S-S bond
• Example 4 Preparation of a fusion gene for the expression of a native antimicrobial peptide and its multimers
• the antimicrobial peptide obtained from the multimers of fusion gene prepared in Example 3 has a homoserine residue at their C-terminus.
• a fusion gene whose sequence was slightly modified from that of the fusion gene prepared in Example 3 and its multimers were prepared as followings:
• Guamerin was used as the acidic peptide
• MSI- 78 GAGKFLKKAKKFGKAFVKILKK-NH 2 : SEQ ID NO:14
• antimicrobial peptide gene I was prepared so that the peptide encoded by this gene may have no methionine residue at the N-terminus, and the C- terminus can be in-frame fused to the following acidic peptide gene in a correct reading frame
• antimicrobial peptide gene II was prepared so that the peptide encoded by this gene may have one methionine residue at N-terminus and peptide synthesis may be terminated at C-terminus.
• antimicrobial peptide genes I and II thus prepared were ligated with an acidic peptide gene, respectively, in the same manner as in Example 3 to prepare fusion genes, and cloned into pBBSl vector digested with Bbsl to construct pBBSl-(GBI) ⁇ and pBBSl-(GB II) i vectors, respectively (see: Figures 4(A) and 4(B)).
• Example 5 Expression and preparation of antimicrobial peptide
• lanes 2 to 14 show cell lysates of E. coli transformed with pET21c, pET21c-B ⁇ , pET21c-B 2 , pET21c-B 4 , pET21c-B 6 , pET21c-
• the peptides were collected by reverse-phase concentration using Sep-Pak, and the antimicrobial peptides with positive charges were purified by QAE-Sephadex (Sigma Chemical Co., USA) anion-exchange chromatography.
• the antimicrobial peptide thus isolated was further purified by reverse-phase HPLC to obtain a pure recombinant antimicrobial peptide.
• the analysis of biological activity of the purified recombinant antimicrobial peptide has revealed that it has the same antimicrobial activity as that of the native one.
• a gene encoding prepromagainin (SEQ ID NO: 15) which has a similar structure to (MB) 6 but contains no cysteine residue, was cloned into a pET21b (Novagen, USA) vector and transformed into E. coli BL21 (DE3) .
• the nucleotide sequence and amino acid sequence translated therefrom are as followings (wherein, the underlined sequence is magainin 1 or magainin 2) :
• lane M shows molecular weight markers (97.4, 66.2, 45, 31, 21.5, 14.4
• lanes 1 and 2 show cell lysates of E. coli transformed with pET21b before and after IPTG induction; lanes 3 and 4 show cell lysate of E. coli transformed with pET21b-
• a fusion gene was constructed with glutathione-S- transferase (GST) sequence and prepro defensin sequence from HNP-I as an acidic peptide gene, and PGQ, as an antimicrobial peptide gene.
• GST glutathione-S- transferase
• the preprodefensin sequence and the GST gene were treated with BspLUllI and Ncol, respectively, and ligated with PGQ gene digested with Ncol.
• One methionine residue was incorporated between the acidec peptide and the antimicrobial peptide for further cleavage with CNBr.
• the obtained fusion genes were cloned into pRSET (Invitrogen, USA) vector and transformed into E. coli HMS174(DE3).
• the expression of the fusion peptides were analyzed by SDS- PAGE ( see. : Figure 6).
• lane M shows molecular weight markers (97.4, 66.2, 45, 31, 21.5, 14.4 Kd)
• lane 1 shows cell lysates of E. coli HMS174(DM3)
• lanes 2 and 3 show cell lysates of E. coli harboring the vectors having the fusion genes of prepro definsin-PGQ and GST-PGQ.
• Example 6 Preparation of a fusion gene comprising a
• Guamerin gene as an acidic peptide gene
• the nucleotide sequence (SEQ ID NO:l) ecoding Guamerin was slightly modified so that its C-terminus can be digested with BspHI, and a methionine codon can be inserted in front of the antimicrobial peptide gene, when the guamerin gene is fused to the antimicrobial peptide gene in order to isolate only a pure antimicrobial peptide by CNBr cleavage of the fusion peptide.
• an N-terminal oligonucleotide containing BamHI and Ndel restriction enzyme sites and a C-terminal oligonucleotide containing BamHI and BspHI restriction enzyme sites were synthesized as followings:
• N-terminal oligonucleotide N-terminal oligonucleotide
• Guamerin-antimicrobial peptide fusion genes were prepared by fusing the synthesized Guamerin gene with various antimicrobial peptide genes shown in Table 1, respectively. That is, the synthesized Guamerin gene was digested with BspHI to give the termini complementary to BspHI or Ncol cleavage site, and fused with the antimicrobial peptide genes synthesized which were digested with Ncol to prepare fusion genes.
• pRSE Invitrogen, USA expression vector was employed.
• the expression vector was digested with BamHI and EcoRI, dephosphorylated, and Guamerin- antimicrobial peptide fusion genes synthesized in Example 6 were cloned.
• E. coli BL21 (DE3) pLysS was transformed with the vectors having fusion genes by CaCl 2 method (see: Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) ) .
• the transformants were cultured in 5ml of LB medium supplemented with ampicillin at 37°C overnight.
• the cultured cells were diluted in 5ml of fresh LB medium in a final concentration of 1% (v/v) , and incubated at 37°C for 2 hours.
• lactose was added to the cultured medium in a final concentration of 2% to induce the expression of the fusion peptides at 37°C for 4 hours.
• the expression of fusion genes was analyzed by SDS-PAGE (see: Figure 7).
• lane M shows molecular weight markers (97.4, 66.2, 45, 31, 21.5, 14.4 Kd) ; lanes 1-8 show cell lysates of E.
• the present invention provides a method for mass production of antimicrobial peptide, which comprises a step of preparing the antimicrobial peptide as a fusion peptide with a foreign peptide.
• the inhibitory effect of the expressed antimicrobial peptide on the growth of host microorganism can be dramatically minimized by fusing it with the acidic peptide. Accordingly, antimicrobial peptides can be produced massively from a recombinant microorganism regardless of the kind of the antimicrobial peptides.
• GCACTCAGCA AAAAAATTTG GAAAAGCTTT TGTGGGAGAG ATAATGAATT 250 CAAAACGAGA TGCAGAAGCA GTAGGACCAG AGGCCTTTGC AGATGAAGAT 300
• CAGAAGCAGT AGGACCAGAG GCCTTTGCAG ATGAAGATTT AGATGAAAGA 450
• MOLECULE TYPE DNA
• ANTI-SENSE NO
• SEQUENCE DESCRIPTION SEQ ID NO: 29:

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