KR100629677B1 - Efficient production method of antimicrobial peptide by fusion with baculoviral polyhedrin in escherichia coli - Google Patents

Abstract

The present invention provides a fusion protein fused to a polyhedral protein derived from a baculovirus and an antibiotic peptide, a gene encoding the same, an expression vector comprising the same, a transformant transformed with the vector, a method for producing the fusion protein, and the It relates to a method for producing antibiotic peptide using a fusion protein. Since the baculovirus polyhedron and halosidine fusion protein of the present invention are expressed in the form of aggregates in E. coli, they can be mass-produced and can be separated and purified by simple manipulation. It can be widely used to produce cheap and effective antibiotics.

Baculovirus, Polyhedral Proteins, Antibiotic Peptides, Escherichia Coli

Description

Efficient production method of antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli}

Figure 1 shows the sea urchin-derived halosidine sequence added to Asn-Gly to be cleaved by hydroxylamine in the 5 'front of the halosidine sequence, and optimized for the main codon of E. coli.

2 is a schematic diagram of a vector pPAP for producing a fusion protein of a polyhedrin of Baculovirus and a desired antibiotic peptide Halocidin.

Figure 3 shows a Western blot using (A) SDS-PAGE and (B) histidine antibody to confirm the formation of aggregates of the fusion protein.

4 shows SDS-PAGE results of fusion proteins obtained by affinity separation and purification.

FIG. 5 shows Tricine SDS-PAGE of the result of each step obtained by cleavage after purification of (A) fusion protein and Tricine SDS-PAGE of the result after cleavage, dialysis and centrifugation of (B) fusion protein.

Figure 6 is the result of measuring the activity of the purified recombinant antibiotic peptide against E. coli.

The present invention provides a fusion protein fused to a polyhedral protein derived from a baculovirus and an antibiotic peptide, a gene encoding the same, an expression vector comprising the same, a transformant transformed with the vector, a method for producing the fusion protein, and the It relates to a method for producing antibiotic peptide using a fusion protein.

All organisms, from microbes to higher organisms, have their own defenses to protect themselves from the harmful environment. In particular, it can be found that this function is enhanced with higher organisms. Anti-antibodies are produced by producing antibiotic peptides, which are self-defense substances that exhibit antimicrobial activity to microorganisms and even mycelium. Prior to the reaction it has been reported to primarily protect itself from external hazards.

Antibiotic peptide is a peptide composed of approximately 50 amino acids, and has a structure in which a hydrophilic portion and a hydrophobic portion are formed by a large number of positively charged amino acids. Therefore, after the antibiotic peptide binds to the cell membrane by the interaction of the positively-charged and negatively-charged cell membranes of the antibiotic peptide, the hydrophobic portion enters the hydrophobic lipid part inside the cell membrane to form a passageway, thereby eliminating permeability inside and outside the cell. In the end, it has been reported to function to disrupt cells. Therefore, existing antibiotics showed a mechanism that prevents the formation of cell membranes, so they are resistant by synthesizing alternative substances that neutralize them, whereas antibiotic peptides target the unchangeable charge characteristics of cell membranes due to the nature of the mechanism of action. The advantage is that it can rule out the problem of resistance to antibiotic peptides. In addition, due to the difference in the membrane structure between the prokaryote (prokaryote) and eukaryote (eukaryote) has the advantage that it only works specifically prokaryotic cells. Therefore, it is currently in the spotlight as an alternative to resistant bacteria, and research is being actively conducted to develop next-generation antibiotics.

Antibiotic peptides were first discovered in insects, amphibians and many vertebrates in the 1980s, since the first discovery of phagocytosis in starfish's body cavity in the 19th century, and is now representative of cecropin, magainin, and diphene. Defensin and the like are commercially available. Baculoviruses / insects, yeasts and Escherichia coli have been used as strains for the production of antibiotic peptides through genetic recombination. All of these have been successful in obtaining recombinant antibiotic peptides, but insect and yeast systems have been commercialized. There was a problem with mass production, the most important factor in the field. Therefore, in order to overcome this problem, an antibiotic peptide was produced in Escherichia coli, and the antibiotic peptide was fused with foreign protein expressed insoluble in Escherichia coli. The purpose of fusion with insoluble foreign proteins is to increase yields, reduce toxicity by the produced recombinant proteins, and to facilitate yield reduction and separation of recombinant proteins by protease. The selection of suitable fusion partners is the most important factor for mass production in E. coli systems.

In the case of producing an antibiotic peptide using E. coli as a strain, although the separation of the recombinant protein was facilitated by fusion with an insoluble foreign protein as described above, the separated protein was treated with a surfactant to treat the foreign protein portion therefrom. In the process of cleaving and separating it from antibiotic peptides, the decrease in yield was more prominent, so the advantages of the E. coli system were not highlighted.

Therefore, in this study, fusion of baculovirus polysaccharides and antibiotic peptides expressed in a neutral environment increased the amount of expression in E. coli and facilitated the separation. By controlling the water solubility of the baculovirus polyhedron as a fusion partner by the change (pH), the separation of the antibiotic peptide and the fusion protein was facilitated, and finally the yield of the antibiotic peptide was increased. In addition, it was confirmed that the recombinant antibiotic peptide produced had the same activity as that of the chemically synthesized peptide against various microorganisms, so that the use of baculovirus polymorphs in the production of antibiotic peptides in Escherichia coli can be used very efficiently. By confirming that the present invention was completed.

An object of the present invention is to provide a fusion protein in which the polyhedral protein and antibiotic peptide of baculovirus are fused.

It is also an object of the present invention to provide a gene encoding the fusion protein.

Still another object of the present invention is to provide a fusion protein expression vector in which a baculovirus polyhedral protein and an antibiotic peptide are fused.

Still another object of the present invention is to provide a transformant transformed with the expression vector. The transformant is preferably E. coli cells.

Still another object of the present invention is to provide a method for producing a fusion protein in which a baculovirus polyhedral protein and an antibiotic peptide are fused by culturing the E. coli transformant to separate the fusion protein.

It is still another object of the present invention to provide a method for preparing an antibiotic peptide comprising the step of separating the antibiotic peptide in the form of the polyhedral protein-antibiotic peptide fusion protein aggregate.

Still another object of the present invention is to provide an antibiotic containing a fusion protein in which a polyhedral protein and an antibiotic peptide of baculovirus are fused or a transformed E. coli cell expressing the fusion protein as an active ingredient.

In order to achieve the above object,

The present invention provides a fusion protein in which a polyhedral protein of baculovirus and an antibiotic peptide are fused. Baculovirus polyhedral proteins have a stable ability in the general natural environment while maintaining the baculovirus in polyhedrin particles to safely preserve the genetic information of the virus in the natural environment. The polygonal particles are composed of a large number, usually 12 unit polyhedral proteins to form a crystal. Recombinant protein in E. coli system is expressed in soluble form or aggregate form depending on the physicochemical properties of the protein and each has advantages and disadvantages. When expressed in soluble form, it generally has the advantage of having biological activity, but is not easy to separate purification and has the disadvantage of being easily attacked by protease. On the other hand, when expressed in the form of an inclusion body, it does not have biological activity, and thus has the disadvantage of performing a refolding process after separation and purification, but it is generally resistant to proteolytic enzymes and easy to separate and purification. Have In particular, aggregate-shaped proteins can be isolated simply with high purity without performing other sophisticated purifications. In particular, when the foreign protein to be produced is harmful to E. coli, which is a host cell, the foreign protein cannot be produced with high efficiency due to the burden placed on the host cell. When a fusion protein fused with a bacteriovirus-derived polyhedral protein and an antibiotic peptide is expressed in E. coli, it can be expressed in the form of aggregates and can be expressed in large quantities without adversely affecting the host cell.

In one embodiment of the present invention, the polyhedral protein derived from the baculovirus is not particularly limited, and may include Autographa californica nuclear polyhedrosis virus (AcNPV), Bombyx mori nuclear polyhedrosis virus (BmNPV), Heliothis zea nuclear polyhedrosis virus (Hz NPV). May contain all of the various polyhedral proteins from different baculoviruses. Among the above-mentioned polyhedral proteins derived from baculovirus, a polyhedral protein derived from AcNPV is particularly preferable. AcNPV specifically works on pests such as Spodoptera exigue , Plutella xylostella and moth Spodoptera litura , Cabbage sting moth ( Trichoplusia ni ) and Pieris rapae do. The genome of AcNPV is about 128kb long and consists of circular double DNA.

In one embodiment of the present invention, the antibiotic peptide is from Halocynthia aurantium , but is not particularly limited thereto, and may include all kinds of antibiotic peptides derived from various organisms. The sea urchin-derived antibiotic peptide is Halocidin.

In one embodiment of the invention, the polyhedral protein site of the fusion protein may preferably have an amino acid sequence as set forth in SEQ ID NO: 4 from AcNPV, and the antibiotic peptide site is preferably from Halocynthia aurantium . All proteins having similarities can be included, including the amino acid sequence set forth in SEQ ID NO: 6.

The present invention also provides a gene encoding a fusion protein in which the polyhedral protein of the baculovirus and an antibiotic peptide are fused. Preferably, the gene of the polyhedral protein portion of the fusion protein of the present invention may have a DNA sequence of SEQ ID NO: 3 derived from AcNPV, the gene of the antibiotic peptide region is from Halocynthia aurantium It may include the sequence of all genes with similar properties, including the DNA sequence of SEQ ID NO: 5.

The present invention also provides a fusion protein expression vector in which a polyhedral protein and an antibiotic peptide of a baculovirus comprising the gene encoding the fusion protein are fused. The expression vector may be any expression vector for bacteria, yeast, animal cells, insect cells and plant cells, as long as it can express the fusion protein of the present invention. As expression vectors for Escherichia coli, pBTrp2, pBTac1, pBTac2 (above Boehringer Mannheim) in addition to pET3, pET11 (above Stratagene), pBAD, pThioHis, pTrcHis (above Invitrogen), pKK223-3, pGEX2T (above Amersham Pharmacia Biotech) ), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol, Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (Stratagene), pTrs30 [Prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [Escarchia coli JM109 / pTrS32 ( Prepared from FERM BP-5408], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), Japanese Laid-Open Patent Publication (Sho) 60-221091], pGKA2 [esquelizia coli IGKA2 (PERM BP- 6798), Japanese Laid-Open Patent Publication No. 60-221091], pTerm2 (US 4686191, US 4939094, US 5160735), pSupex, pUB110, pTP5, pCl94, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia) and pET system (Novagen).

The fusion protein expression vector of the present invention is characterized by having a DNA sequence containing the gene of the fusion protein, preferably may have a DNA sequence comprising the fusion protein gene of SEQ ID NO: 7. Most preferably pPAP (Accession No. KCTC 10577BP: deposited at the Korea Biotechnology Research Institute Gene Bank on January 21, 2005).

The present invention also provides a transformant transformed with the expression vector. The transformant may be any of bacteria, yeast, animal cells, insect cells, and plant cells as long as they can express the fusion protein of the present invention. The transformant is preferably E. coli cells.

In addition, the present invention provides a method for producing a fusion protein fused to a polyhedral protein and antibiotic peptide comprising culturing the E. coli transformant, isolating a fusion protein fused to a baculovirus polyhedral protein and antibiotic peptide to provide. The separation of the fusion protein may be a conventional separation method used for separation of proteins, for example, centrifugation, filtration, salting, and chromatographic methods may be used, and the fusion protein of the polyhedral protein and antibiotic peptide of the present invention is fused. Since it is expressed in the form of intracellular aggregates, it is preferable to easily separate and produce from water-soluble proteins by centrifugation.

In addition, the present invention

(A) culturing the transformed E. coli cells, isolating a fusion protein fused with a polyhedral protein and antibiotic peptide;

(b) cleaving the aggregate of the fusion protein using enzymes or chemicals that specifically cleave the polyhedral moiety or enzymes or chemicals that cleave the boundary region of the polyhedral protein and the antibiotic peptide; And

(c) it provides a method for producing an antibiotic peptide using a polyhedral protein-antibiotic peptide comprising the step of separating the antibiotic peptide using the difference in solubility according to pH.

In step (b), the enzyme that specifically cleaves only the polyhedral moiety or the enzyme that cleaves the boundary region of the polyhedral protein and the antibiotic peptide is preferably an alkaline proteolytic enzyme or chymotrypsin derived from insect heavy solution. It is not limited to this. In particular, a hydroxylamine having the property of recognizing asparagine (Asn) -glycine (Gly) in the protein sequence and cleaving these intermediates may be used to cut the boundary region of the polyhedral protein and the antibiotic peptide. To this end, an asparagine-glycine sequence can be inserted between the polyhedral protein and the antibiotic peptide.

In the step (c), the polyhedral protein is controlled in water solubility by pH change, which is precipitated at neutral pH, so that the cleaved polyhedral protein and the antibiotic peptide can be efficiently separated.

The present invention also provides an antibiotic containing a fusion protein in which a polyhedral protein of the baculovirus and an antibiotic peptide are fused or transformed E. coli cells expressing the fusion protein as an active ingredient.

Antimicrobial experiments were performed using the antibiotic peptides obtained from the recombinant protein of the present invention, and showed the same antibiotic ability as halosidin, an antibiotic peptide derived from chemically synthesized sea urchin (see Table 2).

Antibiotic Activity of Antibiotic Peptides of the Invention Any strain in which the antibiotic peptides of the invention exhibit activity may be included.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention by way of example, the scope of the present invention is not limited to these embodiments.

Example 1: Preparation of fusion protein fused with a polyhedral protein and an antibiotic peptide

<1-1> Expression Vector Preparation of Fusion Protein

In order to insert genes corresponding to polyhedral proteins and antibiotic peptides into the vector, first, a sequence corresponding to BamH I and Bgl II of the pSE420 vector (Invitrogen, USA) was inserted into the pTrcHisC vector (Invitrogen) to prepare a pMPL100 vector. Afterwards, a large amount of the polh gene was amplified by a PCR reaction using a forward primer (SEQ ID NO: 1) and a reverse primer (SEQ ID NO: 2) using the AcNPV baculovirus genome (PharMingen) as a template. The PCR product was inserted into the Nhe I and Bgl II restriction sites of the pMPL100 vector and 68 bp forward and reverse chemically synthesized to encode the desired antibiotic peptide halosidin according to the major codons of Escherichia coli. Oligomers (including cleavage by hydroxylamine and stop codons, Fig. 1) were treated at high temperature to lower their temperature to induce complementary binding to each other, and then placed in place of Bgl II and Pst I of the prepared vector. The inserted and constructed vector was named pPAP (5140bp) (FIG. 2), and the E. coli transformant prepared by introducing the E. coli TOP10 (Invitrogen, USA) was deposited with the Korea Biotechnology Research Institute Gene Bank on January 21, 2005. (Accession No. KCTC 10577BP).

<1-2> Culture of transformants and expression of fusion proteins

The expression vector pPAP obtained in Example <1-1> was introduced into E. coli BL21 (Invitrogen, USA) by a thermal shock method using CaCl ₂ buffer, and an ampicillin-selected transformant was introduced into LB (5 g / L yeast extract, 10 g / L NaCl, 10 g / L tryptone) medium. Expression of the recombinant fusion protein was induced using 1 mM IPTG.

<1-3> Expression of fusion proteins and measurement of expression forms

Confirmation of normal expression of the fusion protein and measurement of its water solubility were performed using SDS-PAGE and Western blot (FIG. 3). SDS-PAGE and Western blot analysis were performed by the following method. Add 100 μl of buffer for SDS-PAGE (0.5M Tris-HCl (pH 6.8), 10% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.25% bromophenol blue) and boil at 100 ° C. for 5 minutes. Denatured. The sample prepared as described above was subjected to electrophoresis on 15% SDS-polyacrylamide gel, and then transferred to a nitrocellulose membrane under 15V voltage and subjected to color reaction using histidine protein antibody (Santa Cruz, CA, USA).

SDS-PAGE was performed after incubation for 5 hours with and without expression-induced cells to confirm expression, and specific proteins were detected only in cells with expression-induced expression near 33 kDa. In order to obtain the same result as a result of Western blot using histidine antibody, it was confirmed that the desired fusion protein was normally expressed.

In addition, in order to confirm whether the fusion protein is expressed in the form of an inclusion body in E. coli, the E. coli cells were subjected to an analysis of the cell disrupted precipitate sample and the supernatant sample by centrifugation after ultrasonic grinding. The fusion protein band of the present invention was detected, but not in the supernatant sample. Therefore, the fusion protein of the present invention was found to be expressed in the form of insoluble aggregates in E. coli.

Example 2: Chemical Synthesis of Control Antibiotic Peptides

The same antibiotic peptide was then synthesized using an automated solid phase peptide synthesizer (Pioneer; Applied Biosystems) as a control for the produced recombinant antibiotic peptide, and then purified using reversed phase high pressure liquid chromatography.

Example 3: Isolation of Fusion Proteins

Polygon-antibiotic peptide of the present invention is expressed in the form of aggregates in Escherichia coli as confirmed in Example <1-3>, and is separated from the aggregates because the supernatant and cell debris are present in the fragments when the centrifuge separates It could be separated using the method.

Specifically, E. coli cells were recovered by centrifuging the culture solution at 4 ° C and 5500 rpm for 15 minutes, and 3 ml of cell disruption buffer (50 mM Tris-Cl, pH 8.0; 1 mM EDTA, pH 8.0; 100) per cell weight. mM NaCl) was added to gently resuspend the cells. Then at 4 ° C. add 4 μl of 100 mM PMSF (Protease Activity Inhibitor (Sigma)) and 80 μl of 10 mg / ml lysozyme per cell weight and stir gently for about 20 minutes, 4 mg per cell weight Deoxycholic acid was added and left in the incubator at 37 ° C. until no longer sticky (if the viscosity increased, 20 μg of 1 mg / ml DNase I was added). Thereafter, the cell solution was pulverized with a high pressure pulverizer (Microfluidizer M-110Y; Microfluidics) at a pressure of 1000 bar at a flow rate of 450 ml / min, and then centrifuged at 14,000 rpm and 4 ° C. for 30 minutes. A pellet containing only and insoluble protein was obtained. The precipitate was further dissolved in a dissolution buffer (100 mM NaH ₂ PO 4, 8 M urea, 10 mM Tris-Cl; pH 8.0) to selectively dissolve only insoluble protein in the precipitate, followed by centrifugation at 15,000 rpm to separate only the supernatant.

Ni-nitrilotriacetate-agarose resin (Ni ²⁺ -nitrilotriacetate-agarose resin, Qiagen) and elution buffer (250 mM imidazole, 100 mM NaH ₂ PO 4, 8 M urea, 10) to obtain higher purity fusion proteins After performing friendly separation purification using mM Tris-Cl; pH 5.9), dialysis under alkaline conditions (10 mM Tris-Cl; pH12), lyophilization, dissolving it in distilled water and analyzing SDS-PAGE, image analysis software Quantification was performed using (Gel-Pro Analyzer, Media Cybernetics, USA).

As a result, 86% of high purity fusion protein was isolated from the initial cell lysate (FIG. 4).

Example 4 Cleavage of Polyhedral-Antibiotic Peptides Using Specific Chemical Reagents

In order to isolate only antibiotic peptides having anti-biological properties among the fusion proteins, an experiment was performed in which the boundary portion of the baculovirus polyhedral protein and the antibiotic peptide was treated with a chemical reagent, hydroxylamine.

The hydroxylamine is a reagent that recognizes asparagine (Asn) -glycine (Gly) in the protein sequence and cleaves these intermediates, and between the baculovirus polyhedron and the antibiotic peptide to induce cleavage by hydroxylamine. An asparagine-glycine sequence was inserted into it (see FIG. 1).

For cleavage experiments, three times the volume of cleavage buffer (0.22 M trizma base, 1.7 M hydroxylamine-HCl, 4.5 M guanidium-HCl, 1% 1-propanol) in the fusion protein solution isolated in Example 3; pH8.8) was added, followed by reaction at 55 ° C. for one day. After cleavage, Tricine SDS-PAGE analysis showed no bands of the fusion protein of the polyhedral-antibiotic peptide, and the band of the peptide type having a few kDa and a band smaller in size than the fusion protein appeared simultaneously. Therefore, it was confirmed whether the fusion protein was cleaved by hydroxylamine, and the efficiency was also confirmed to be very high.

Samples subjected to cleavage were neutral buffer (20 mM PBS; pH7.4) to remove guanidium-HCl, a surfactant that enhances the solubility of hydroxyl amines and baculovirus polyaggregates that are toxic to cells. As a result of dialysis, it was possible to observe the mass precipitation phenomenon of the protein (Fig. 5. B). Therefore, as a result of Tricine SDS-PAGE analysis of the supernatant through centrifugation, it was confirmed that the baculovirus polyhedron and some degradation fragments thereof were removed (FIG. 5A). As a result, it means that the polyhedral protein is precipitated at neutral pH in the dialysis process of removing the surfactant, which shows that the separation of the fusion partner can be easily performed with high efficiency by simple adjustment.

Example 5: Purification of Recombinant Antibiotic Peptides

The desired recombinant protein was obtained from Example 4, but the purity of the resultant product (25%, Table 1) was low, and purification was performed using a method of reversed phase high-pressure liquid chromatography to increase the purity.

The sample contained 3 ml / min in a tube (4.6 × 100 mm Chromolith performance RP18e column, Merck) containing 0.1% (volume / volume) trifluoroacetic acid and acetonitrile with a linear gradient of 20 to 40%. Injected at a flow rate for 1 hour, and observed at 215 nm absorbance to obtain the desired recombinant antibiotic peptide, the analysis was performed via Tricine SDS-PAGE.

As a result, a single peptide band was observed on the polyacrylamide gel and confirmed to be consistent with the size of the same antibiotic peptide chemically synthesized (FIG. 5. A). In addition, as a result of quantitative analysis using image analysis software, it was confirmed that a recombinant antibiotic peptide having a final yield of 33% and a purity of 90% or more was obtained (Table 1).

Isolation and Purification of Recombinant Antibiotic Peptide Hal18 step Total protein (mg) Hal18 Volume (mg) Yield (%) Purity (%) Affinity Separation 30 1.56 100 5 Cutting, dialysis, centrifugation 3 0.75 48 25 RP-HPLC separation 0.56 0.51 33 91

Example 6: Antimicrobial Activity of Recombinant Antibiotic Peptides

In order to confirm the antimicrobial activity of the recombinant antibiotic peptides obtained in Examples 4 and 5, the minimum inhibitory concentration (minimal) against Gram-negative bacteria ( E. coli Top 10) and Gram-positive bacteria ( S. aureus ATCC 6538p) Inhibition concentration and radial diffusion assay were performed.

The two microorganisms were incubated in 3% TSB medium (3% tryptic soy broth; Difco) for 0.5 days for radius diffusion analysis, and then 1: 1000 was transferred to fresh TSB medium again at 37 ° C for 2.5 hours. The cell culture solution obtained by incubating at 250 rpm was adjusted to OD ₆₀₀ = 0.3, and 1% of this was PBS containing 3% TSB preheated to 42 ° C and 1% (weight / volume) of agarose (pH 7.4). After addition to the buffer, the Petri dishes were thinly and uniformly poured and hardened. Inject 7 holes of 3 mm diameter into the hard gel and inject 10 μl of samples for activity check into each hole and incubate for 3 hours at 37 ° C., followed by further addition of 10 ml of 6% TSB agarose medium. The diameter of the circular clean region produced by incubating at 12 ° C. for 12 hours was measured. As a result, it was observed that the recombinant antibiotic peptide before purification isolated from Example 4 with respect to the Gram negative group Escherichia coli forms a clean zone similar to the same antibiotic peptide chemically synthesized as a control (Fig. 6). Therefore, this result proved that the antimicrobial activity of the recombinant antibiotic peptide produced by the method of the present study was restored, and the same result was confirmed for Gram-positive Staphylococcus aureus.

In order to measure the minimum inhibitory concentration, a certain concentration of the sample obtained in Example 5 was mixed with an equal volume of cell culture medium having a cell concentration of 10 ⁵ cfu / ml, and then cultured in a 96-well plate at 37 ° C. for 12 hours. As a result of measuring the minimum concentration of antibiotic peptide samples in which microorganisms do not grow at all, the minimum inhibitory concentrations of Escherichia coli and Staphylococcus aureus were 40 µg / ml for the samples of low purity obtained as a result of dialysis after cleavage of the fusion protein. 20 µg / ml, but the samples purified by reversed phase-high pressure liquid chromatography were measured to be 10 µg / ml and 2.5 µg / ml, respectively (Table 2). Therefore, the antibiotic peptide used in this study showed higher activity on Gram-positive bacteria than Gram-negative bacteria. In addition, in the case of purified samples, since the chemically synthesized control antibiotic peptide and the same value for both microorganisms, this proves that the antibiotic peptide having the same activity can be obtained even if the recombinant antibiotic peptide is produced by the method of the present invention. It is.

Determination of Minimum Inhibitory Concentration (㎍ / ml) of Recombinant Antibiotic Peptide Hal18 Escherichia coli Staphylococcus aureus Recombinant Hal18 obtained after dialysis and centrifugation after affinity separation 40 20 RP-HPLC Purified Recombinant Hal18 10 2.5 Chemically synthesized Hal18 10 2.5

The polyhedral-halosidine fusion protein in which the polyhedral protein and the antibiotic peptide of the baculovirus of the present invention are expressed in the form of aggregates in Escherichia coli can be mass-produced by reducing the burden of the host cell, and the baculovirus polyhedron. Due to the specific dissolution and precipitation phenomenon according to the pH, the separation and purification is simple, and even after removing the fusion partner through chemical reagents, no additional refolding process is required, thus maintaining the same antimicrobial activity as the control group. It can be widely used for the production of peptides.

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Claims (13)

A fusion protein in which a polyhedral protein of baculovirus and an antibiotic peptide having the amino acid sequence of SEQ ID NO: 6 are fused. The fusion protein of claim 1, wherein the baculovirus is selected from the group consisting of Autographa californica nuclear polyhedrosis virus (AcNPV), Bombyx mori nuclear polyhedrosis virus (BmNPV), and Heliothis zea nuclear polyhedrosis virus (HzNPV). . The fusion protein of claim 1, wherein the antibiotic peptide is derived from Halocynthia aurantium . The fusion protein of claim 1, wherein the polyhedral protein portion of the fusion protein has an amino acid sequence represented by SEQ ID NO: 4. delete The gene encoding the fusion protein of claim 1. The gene encoding the fusion protein according to claim 6, wherein the gene of the polyhedral protein portion of the fusion protein has a DNA sequence of SEQ ID NO: 3. The gene encoding a fusion protein according to claim 6, wherein the gene of the antibiotic peptide region of the fusion protein has a DNA sequence of SEQ ID NO: 5. A fusion protein expression vector in which a baculovirus polyhedral protein comprising the gene of any one of claims 6 to 8 and an antibiotic peptide are fused together. 10. The expression vector according to claim 9, wherein the fusion protein expression vector is pPAP comprising a DNA sequence of SEQ ID NO. E. coli transformants transformed with the expression vector of claim 9. A method for producing a fusion protein in which a polyhedral protein and an antibiotic peptide are fused by culturing the E. coli transformant of claim 11, and separating the fusion protein in which the baculovirus polyhedral protein and the antibiotic peptide are fused. (a) culturing the transformed Escherichia coli cell of claim 11, isolating a fusion protein in which the polyhedral protein and the antibiotic peptide are fused; (b) cleaving the aggregate of the fusion protein using enzymes or chemicals that specifically cleave the polyhedral moiety or enzymes or chemicals that cleave the boundary region of the polyhedral protein and the antibiotic peptide; And (C) a method for producing an antibiotic peptide using a polyhedral protein-antibiotic peptide comprising the step of separating the antibiotic peptide using the difference in solubility according to pH.

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