KR100230959B1 - Preparation method using antifungal protein and recombinant microorganism isolated from brown larva - Google Patents

Abstract

The present invention relates to an antifungal protein isolated from brown larva larvae and a method for producing the same using recombinant microorganisms. More specifically, the present invention provides a novel antifungal protein (Tenecin 3) isolated from brown larvae belonging to the beetle neck, a gene encoding the same, an electric gene and an expression vector capable of producing antifungal protein (Tenecin 3), The present invention relates to a method for producing a large amount of recombinant antifungal protein using an electric expression vector and a variant of the electric antifungal protein (Tenecin 3). According to the present invention, the novel antifungal protein Tetesin 3 and its variants isolated from brown larvae are produced in large quantities by genetic engineering methods. This mass-produced Tennessine 3 and its variants can be used in pharmaceutical compositions of antifungal agents with pharmaceutically acceptable ingredients.

Description

Antifungal protein and recombinant microorganisms isolated from brown larvae

The present invention relates to an antifungal protein isolated from a brown rice oil layer and a method for producing the same using a recombinant microorganism. More specifically, the present invention provides a novel antifungal protein (Tenecin 3) isolated from brown larvae belonging to the beetle neck, a gene encoding the same, an electric gene and an expression vector capable of producing antifungal protein (Tenecin 3), The present invention relates to a method for producing a large amount of recombinant antifungal protein using an electric expression vector and a variant of the electric antifungal protein (Tenecin 3).

It is well known that insects are one of the animals that have evolved successfully. An important factor in this successful evolution is the development of insect defense systems, which not only provide sufficient protection against infection, but also enable insects to have a diverse range of life. Therefore, insects must have various immune defense strategies and specificities. Because of this possibility, insects have long been used in immune defense research.

In insects, the first defense is the physical barrier provided by the epidermis, which is a passive defense. The second defense is active defense, including cellular response and humoral response. An example of this active defense is when the epidermis is injured, exposing epithelial cells or internal tissues to which bacteria are infected. For cellular responses to these bacterial infections, hemosite, a type of insect blood (hemoly mph) cell, is mobilized. This hemosite acts to phagocytosis or trap nodules. On the other hand, the humoral response is induced or increased the production of a variety of blood lymphatic proteins in response to wounds and infection. Many of these hemolymph proteins are antibacterial proteins that can provide a strong defense against bacteria. In addition, hemostatic action is activated to repair wounds and prevent further bacteria from entering.

Many antibacterial proteins have been found in hemoglobin of insects, and research on them has been carried out, but relatively few studies on antifungal proteins have progressed. Indeed, cecropin and defensin family antibacterial proteins have been reported to have antifungal activity, but there are no reports on proteins having only antifungal activity without antibacterial activity.

Therefore, the present inventors have diligently researched to find a protein having only antifungal activity in insects and, for the first time, isolated new antifungal protein (Tenecin 3) from brown larvae belonging to the beetle neck and revealed its characteristics. The gene encoding the anti-fungal protein (Tenecin 3) is isolated from the larval larvae, and it was confirmed that the recombinant anti-fungal protein (Tenecin 3) can be produced in large quantities, thereby completing the present invention. In addition, it was found that even when the amino acid was deleted or substituted for the antifungal protein (Tenecin 3), a strain was maintained in which the antifungal activity was maintained.

After all, the first object of the present invention is to provide the amino acid sequence of the antifungal protein (Tenecin 3) isolated from brown larva larvae.

A second object of the present invention is to provide a nucleotide sequence of a gene encoding an electric antifungal protein (Tenecin 3).

A third object of the present invention is to provide an expression vector containing the gene of the antifungal protein (Tenecin 3) and capable of producing antifungal protein.

The fourth object of the present invention is to provide a method for producing a large amount of recombinant antifungal protein (Tenecin 3) using the electric expression vector.

A fifth object of the present invention is to provide a variant of the electroantifungal protein (Tenecin 3).

1 shows some amino acid sequences of antifungal protein (Tenecin 3).

Figure 2 shows the entire amino acid sequence of the antifungal protein (Tenecin 3) and its coding sequence.

3 shows a genetic map of the plasmid encoding the maltose fusion protein.

4 is a schematic diagram showing the manufacturing process of a plasmid expressing an intact antifungal protein (Tenecin 3).

5 is a diagram showing an antifungal protein (Tenecin 3) variant of the N-terminal or C-terminal deletion using His-tag fusion antifungal protein (Tenecin 3).

FIG. 6 shows a variant of an antifungal protein (Tenecin 3) obtained by in situ alanine mutation of the C-terminal 33 amino acid portion of the antifungal protein (Tenecin 3).

Figure 7a is a graph showing the growth inhibitory effect of Saccharomyces cerevisiae according to the treatment concentration of crude antifungal protein (crude Tenecin 3) obtained from brown larva larvae.

Figure 7b is a graph showing the growth inhibitory effect of Candida albicans according to the treatment concentration of crude antifungal protein (crude Tenecin 3) obtained from brown larva larvae.

8 is a graph showing the growth inhibitory effect of the crude antifungal protein (crude Tenecin 3) obtained from brown larva larvae against filamentous fungi.

9 is a graph showing the growth inhibitory effect of crude antifungal protein (crude Tenecin 3) obtained from brown larva larvae against Fusarium oxysporum.

10 is a graph showing the effect of crude antifungal protein (crude Tenecin 3) obtained from brown larvae larvae on the growth of yeast fungi under the condition that the growth was stopped.

11 is a graph showing the effect of the fusion protein of maltose binding protein-antifungal protein (Tenecin 3) on the growth of Can dida albicans.

12 is a graph showing the effect of the fusion protein of maltose binding protein-antifungal protein (Tenecin 3) on the growth of Cryptococcus neoformans.

Figure 13 is a graph showing the effect of the fusion protein of maltose binding protein-antifungal protein (Tenecin 3) on the growth of Aspergillus nidulans.

14 is a graph showing the effect of the fusion protein of maltose binding protein-antifungal protein (Tenecin 3) and its cleavage product by factor Xa on the growth of Candida albicans.

15 is a graph showing the antifungal activity of N-terminal or C-terminal deletion variants of the antifungal protein (Tenecin 3).

FIG. 16 is a graph showing the antifungal activity possessed by specific site mutation variants of the antifungal protein (Tenecin 3).

Hereinafter, the present invention will be described in more detail.

Antifungal protein is a renal function protein, so it is not only the academic value to understand biophenomena, especially immune action, but the effective antifungal antibiotics have not been developed through the identification of protein itself, biosynthetic process and antifungal mechanism of action. It has a practical value that can be used as an important biological material in the industry. In order to develop new functional biological materials, the tasks to be performed are not only to isolate, purify, and characterize them, but also to study in various fields such as how they are synthesized in vivo, their gene expression mechanisms, and the mechanisms in which they work. It should be complementary and systematic. In particular, when new functional biomaterials are present in trace amounts in nature, research on biosynthesis processes and gene expression mechanisms is very necessary and important when attempting to supply them using genetic recombination methods. In addition, the results of studies of protein properties and mechanisms of action may enable the creation of highly functional new biomaterials.

The results from these studies suggest that antifungal proteins can be used directly as renal functional biomaterials and, as a leading material, are expected to be effective in creating new biomaterials, as well as cell defense mechanisms against external invasion. Expectations from the academic aspect of identifying new biological phenomena in the field of immune action are also great. Therefore, the results obtained through the present invention can be utilized for the development of new drugs of antifungal agents as well as academic development. In this regard, the present invention is a good example of efficiently carrying out the development of new functional biological material through basic research, avoiding the conventional preconceived notion that basic research and practical use are separate. In this context, the present invention will be described in more detail as follows.

In order to find out the amino acid sequence for a novel antifungal protein (hereinafter referred to as 'Tenecin 3') and the cDNA sequence encoding it in brown larva larvae, firstly from the brown larva larvae Pure was separated. That is, the supernatant obtained by injuring the brown larva larvae, sieving and centrifuging the body fluid was heat-treated at 100 ° C. to remove the polymer protein, and then subjected to C ₁₈ reverse phase open column chromatography in primary and secondary phases, 1 Tennessine 3 was purified purely by C ₁₈ reverse phase HPLC and gel filtration HPLC over both primary and secondary. The amino acid sequence of Tennessine 3 thus separated was determined.

In addition, cDNA library was prepared by separating mRNA from the total cell RNA of the brown larva larvae, and the cDNA of tennessine 3 was cloned by polymerase chain reaction (PCR), and its nucleotide sequence was determined.

The cloned Tennessine 3 cDNA gene is inserted into a protein expression function plasmid to produce Tennessine 3, or a vector to which a fusion partner is linked, for example, a MBP (maltose binding protein) gene, to facilitate purification of Tennessine 3 The vector or histidine-labeled Tennessine 3 is inserted into a vector which can then be introduced into the microorganism and cultured to induce the expression of Tennessine 3, thereby obtaining the fusion partner-Tenesin 3 Can produce a fusion protein.

On the other hand, to find a strain of Tennessine 3 with antifungal activity, a mutation that deletes the N-terminal or C-terminus of mature Tennessine 3 without a signal peptide, or a specific site mutation that induces substitution with alanine , Amino acids 94-96, amino acids 70-96, amino acids 61-96, amino acids 48-95, amino acids 19-28, 19 based on the amino acid sequence of Tennessine 3 including a signal peptide Amino acids 67-70 based on amino acid sequence of Tennessine 3, including amino acid number 35 to amino acid, amino acid number 19-47, amino acid number 19-55, amino acid number 19-63 deleted, and signal peptide It was confirmed that the amino acid substitution of amino acids 75-77, amino acids 77-80, amino acids 82-85, amino acids 86-89, amino acids 91-94 with alanine had antifungal activity.

In addition, the gene encoding the above-described strain of Tennessine 3 is prepared, inserted into the expression vector, then introduced into the microorganism and cultured, and the expression of the strain of Tennessine 3 is induced by the process of obtaining Tennessine 3 Can produce large quantities.

Tennessine 3 and its variants, either directly isolated from brown larvae or made from recombinant microorganisms, are mutated in mutagenic fungi, such as Candida albicans (causes the oral cavity and digestive tract) and Cryptococcus neoformans (lungs and meninges). Induces growth). Thus, mass-produced Tennessine 3 and its variants can be used in pharmaceutical compositions of antifungal agents with pharmaceutically acceptable ingredients.

Meanwhile, in the amino acid sequence of the present invention, "functionally equivalent" means Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And all proteins substituted with a combination such as Phe, Tyr. In addition, in the nucleotide sequence of the present invention, the term "functional equivalent" refers to all genes having a nucleotide sequence capable of providing the amino acid of the above combination in the nucleotide sequence of the Tennessine 3 gene.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to specifically describe the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1

Pure Separation of Antifungal Proteins from Brown Peel Larvaes and Their Amino Acid Sequence Determination

Antifungal protein isolation from brown larva larvae and amino acid sequencing of the protein were carried out by the detailed method described below.

(1) Antifungal force measurement method

First, the composition of the medium to be used, the type and composition of the insect saline (insect saline) and the various buffers (buffer) used in the PAGE is as follows, and protein quantification in each step by the micro-Lowry method Isolated and purified protein was quantified.

Composition of Solid Medium for Candida albicans Culture

4g yeast extract

3.4 g of glucose

5.2 g agar

Nitrofurazon 0.2g

DDW (double distilled water) 400ml

Adjusted to pH 6.0 with HCl, sealed and stored at 4 ° C. (HCl diluted with water was used).

Composition of Sabouraud Broth for Candida albicans Culture

3 g of glucose

2 g of pepron

DDW 200ml

After adjusting to pH 5.8 with HCl sterilization was used.

Composition of 1X Insect Saline

130 mM NaCl 0.759 g

5 mM KCl 0.037 g

1 mM CaCl ₂ 2H ₂ O 0.014 g

DDW up to 100ml

After adjusting to pH 6.0, sterilized and refrigerated.

Composition of Loading Buffer for Sample Injection in Acid-urea PAGE

1 M HCl 1ml

0.5ml 2-mercaptoethanol

Urea 5.4g

0.5 ml of pyronin Y

DDW up to 10ml

After preparation it was stored at -20 ℃.

Composition of running buffer for sample development on acid-urea PAGE

0.9 M acetic acid (pH 3) 51.5 ml

DDW up to 1000ml

After preparation, put in a brown bottle and stored at 4 ℃.

Composition of buffer for sample injection during SDS-PAGE

0.5M Tris-HCl (pH 6.8) 3.75 ml

20% SDS 1.5ml

2-mercaptoethanol 0.6ml

Glycerol 2.65ml

0.1% BPB 1.5ml

DDW up to 10ml

Buffer for 5X Sample Development on SDS-PAGE

Tris base 45g

Glycine 140g

SDS 5g

DDW up to 1000ml

On the other hand, the antifungal protein activity in the following Examples was measured by the following colony count (colony count) and spectroscopy (spectroscopy).

Colony counting

Candida albicans (ATCC 36232) was inoculated in Sabouraud broth, incubated for 16 hours at 37 ° C, and then 1 ml of the solution was centrifuged at 8,000 rpm (4 ° C) for 5 minutes. Then, the bacteria were suspended in insect saline, and some of the solution was taken to measure the absorbance at 650 nm and adjusted to about 0.3 (2 × 10 ⁶ / ml). Then, 50ul of this solution was placed in a test tube and immediately collected. 50ul of insect body fluids were removed. Then, shake culture for 2 hours at 37 ° C, take 50ul of this solution diluted 100-fold with insect saline, spread on medium for cultivation of Candida albicans, incubate at 37 ° C for 48 hours, and count the resulting colony water. Antifungal activity against the stock solution was evaluated.

Spectrophotometry

Candida albicans (ATCC 36232) was incubated for 24 hours in a solid medium, one colony was collected and incubated for 16 hours at 37 ° C. on a liquid medium. The culture solution was centrifuged at 8,000 rpm, and the residue was suspended in insect saline. A portion of the solution was adjusted so that the absorbance at 650 nm was about 0.3 (2 × 10 ⁶ cells / ml). Take a fixed amount of this solution and take 200ul of the solution diluted 10-fold with liquid medium, add a certain amount of protein of each fraction separated by column chromatography, shake-incubate at 37 ℃ for 12 hours, and measure the absorbance at 650nm. Antifungal activity was evaluated by determining the growth of Candida albicans.

(2) collecting fluid from brown larva

Larvae (4,000 to 5,000 larvae) of brown mealworms (4,000 to 5,000) were wounded with needles, and fluids were collected, and then centrifuged at 20,000 rpm and 4 ° C to separate blood lymph and hemosite. Taken and stored until use at -80 ° C. The body fluid of this stock solution was used to measure antifungal activity by colony counting or to separate and purify antifungal protein.

(3) Isolation, Purification and Characterization of Antifungal Proteins

In order to investigate the biochemical properties of proteins having antifungal activity by separating and purifying them into a single substance, first, purification of the electrical proteins was performed in the following order.

① heat treatment

In order to remove the polymer protein contained in the body fluids of the insects measured antifungal force by colony counting method for the body fluids of the insects collected in the above (2), heat treatment was performed as follows. The body fluid was diluted 10-fold with distilled water in a 50 ml plastic tube, and then heat-treated for 15 minutes in 100 ° C of cut water and left at 4 ° C for 24 hours. Subsequently, centrifugation was performed at 3,000 rpm, and only the supernatant was carefully taken and used for the separation and purification of the antifungal protein of the next step.

② First C ₁₈ Reversed Phase Chromatography

In order to purify the antifungal protein from the collected liquid to the heat treatment, in which C ₁₈ equilibrated with water containing 5% TFA reverse phase column; infusion for _{(C 18 reverse phase open column,} 0.7 × 30cm Waters Co., USA) , and the linear density Absorbance at 280 nm was measured by eluting with a gradient (A: 5% CH ₃ CN in 0.05% TFA, B: 45% CH ₃ CN in 0.05% TFA) to determine the antifungal activity against each fractional part (15 ml) with absorbance. Was measured.

In order to investigate the biochemical properties of proteins having antifungal activity by separating and purifying them into a single substance, first, purification of the electrical proteins was performed in the following order.

③ Second C ₁₈ open phase chromatography

Fractions showing antifungal activity in the first C ₁₈ reversed phase column chromatography were combined and subjected to second reversed phase column chromatography. At this time, the column and the elution solvent used were the same as in the first time.

④ Primary C ₁₈ Reversed Phase HPLC

Fractions showing antifungal activity in the second C ₁₈ reversed phase column chromatography were collected and injected into a C ₁₈ reversed phase HPLC column (Toyosoda, JAPAN, ODS 12OT, 0.5 × 30 cm), and a linear concentration gradient (flow rate: 1 ml / min, A: 5% CHCN ₃ in0.05% TFA, B: 90% CHCN ₃ in 0.05% TFA), the antifungal force was measured for each fraction and only the active fraction was fractionated.

⑤ Secondary C ₁₈ reversed phase HPLC

The fractions showing the antifungal activity in the primary C ₁₈ reverse phase HPLC was collected HPLC car 2 C ₁₈ reverse phase. In this case, the column used was C ₁₈ reverse phase HPLC (Synchropack, 0.5 × 30 cm, Synchrion Co., USA), and the linear concentration gradient used was different from that of the first C ₁₈ reverse phase HPLC except that the elution time was lengthened. The same was done. Antifungal activity was measured for each fraction obtained by elution, and only the active fraction was aliquoted and the protein was quantified, and its purity was examined by SDS-PAGE and acid-urea PAGE.

⑥ Gel Filtration HPLC

In order to purify the amino acid sequence of the antifungal protein and a single substance for investigating its properties, a gel filtration HPLC column (Toyosoda, JAPAN, TSK gel G3000SW, 1 × 30cm) was collected by collecting the fractions showing antifungal activity in the second C ₁₈ reversed phase HPLC. ), And separated by an isocratic system (flow rate: 0.5ml / min, 30% CH ₃ CN in 0.05% TFA), the antifungal force measurement and protein quantification for each fraction.

⑦ Measurement of molecular weight and purity of antifungal protein by SDS-PAGE and acid-urea PAGE

In order to determine the amino acid sequence of the antifungal protein fractions collected by the ⑤ and ⑥ HPLC, it is necessary to confirm whether the protein is purely purified into a single substance, and to confirm the molecular weight of the protein, SDS-PAGE and Acid-urea PAGE was performed. SDS-PAGE was electrophoresed using a 20% separating gel and a 3.75% stacking gel, stained with Commassie Brilliant Blue G-250, and then standard protein. The size and purity of the antimicrobial protein was confirmed by comparison with the band.

(4) Determination of amino acid sequence of isolated antifungal protein

① Reduction and alkylation of proteins for amino acid sequencing

When disulfide bonds by cysteine residues are present in the antifungal protein, fragmentation of the protein by endopeptidase treatment is not easy, so disulfide bonds are reduced, alkylated, and then endopeptidase treated. That is, 30 ug of the purified antifungal protein was dissolved in 150ul of solution (6M guanidine-HCl in 0.25M Tris buffer, pH 8.5), 7.5ul 2-mercaptoethanol was added, and nitrogen or argon gas was injected. Subsequently, the mixture was reacted for 2 hours under light shielding conditions, 6ul of 4-vinylpyridine was added, and then left at room temperature for 2 hours to separate and purify the alkylated protein by HPLC, followed by endopeptidase treatment. . At this time, the column used was ABI Aquapore RP-300 column.

② fragmentation of antifungal protein with Lysylendopeptidase

In order to determine the partial amino acid sequence of the antifungal protein identified as a single substance in (3) -⑦ and the antifungal protein fragmented in ① above, lysine-specific endopeptidase was added to 1 nmol of the antifungal protein in 50 mM Tris-HCl (pH 9.0) solution. And added to a concentration of 0.025ug / ul, and incubated at 30 ° C for 19 hours, and then the reaction solution was poured into a C ₁₈ reverse phase column (Synchrion, INC., Synchropack, 0.5 × 30 cm), and a linear concentration gradient (A: 0 Peptide fragmented with% CH ₃ CN in 0.05% TFA, B: 100% CH ₃ CN in 0.05% TFA). Peptide fragments thus obtained were Speed-Vac dried, and then partial amino acid sequences were determined by an automatic amino acid sequencer (see FIG. 1).

③ trypsin, Asp-N and V8 endopeptidase treatment

In order to determine the whole amino acid sequence while fragmenting the antifungal protein with lysine endopeptidase in ②, the other endopeptidase treatment was also required. By comparing the amino acid sequences of the obtained fragments, it was possible to determine the entire amino acid sequence from the N-terminus with overlapping amino acids (see Fig. 2).

First, in the trypsin treatment, 1 nmol of protein was dissolved in 40ul of 0.2M NH ₄ HCO _3, 0.33ug of trypsin was added thereto, followed by incubation at 37 ° C for 14 hours, followed by heat treatment at 100 ° C for 2 minutes to separate and purify the protein fragments by HPLC. Asp-N endopeptidase treatment was also dissolved 1nmol of protein in 50ul of 50mM phosphate buffer (pH 8.0), 0.5ug proteolytic enzyme was added and incubated for 18 hours at 37 ℃, separated and purified by HPLC. , V8 endopeptidase treatment was dissolved 1nmol protein in 200ul of 0.1M ammonium acetate (pH 4.0), 0.3ug proteolytic enzyme was added and incubated for 18 hours at 37 ℃, then separated and purified by HPLC. In the above, the column and the eluting solvent of the HPLC used were the same as that used in the above ②.

Example 2

CDNA sequencing of antifungal proteins

Total cell RNA was extracted from larvae of larvae (T. molitor) and 5 ul reverse transcriptase buffer 5 ul, 20 mM dNTP 1 ul, RNase inhibitor 1 ul, 25 mM MgCl _{2 2} ul, synthesized 50 mM oligo (dT) ₁₈ primer and 1 μl of M-MuLV (Moloney Murine Leukemia Virus) reverse transcriptase (NEB) was added, the total volume was adjusted to 20 μl with tertiary distilled water, and then reacted at 37 ° C. for 1 hour and 30 minutes to prepare a first chain cDNA. In addition, cDNA was prepared by separating mRNA from the electric total cell RNA and cloned into lambdagtll vector to prepare cDNA library.

Using the first chain cDNA and cDNA library prepared above as a template, the cDNA of the antifungal protein (Tenecin 3) was cloned by PCR (polymerase chain reaction) method. At this time. PCR conditions were set at a denaturation temperature of 94 ° C. and an extension temperature of 72 ° C., and the annealing temperature was appropriately adjusted according to the Tm value of the primers used in each PCR. A total of 35 cycles were performed. Initially, the sample was left at 72 ° C. for 10 seconds, and was left at 120 ° C. for 120 seconds at 120 ° C., 120 seconds at the binding temperature, and 120 seconds at 72 ° C. for 2 cycles, and 94 times thereafter. 45 seconds at ℃, 60 seconds at the binding temperature, 60 seconds at 72 ℃. The last extension time was 300 seconds. The PCR reaction was 100ul in total, 1ul of running DNA, 1ul of 50μM 5 'and 3' primers, 10x Taq polymerase buffer solution (100mM Tris-Cl (pH 8.3), 500mM KCl, 0.1% gelatin) 10ul, 20mM dNTP 1ul , 25mM MgCl ₂ 8ul, and 1q Taq DNA polymerase (Bionia, Korea) were added, and water was added to make 100ul.

The cDNA thus obtained was subcloned into phagemid to determine the base sequence of the antifungal protein (Tenecin 3) using the Sequenase Version 2.0 kit (United States Biochemical, USA) by Sanger's method. 2 degrees). In Figure 2, the dark italics indicate signal peptides.

Example 3

Mass Production of Tennessine 3 Using Maltose Binding Protein (MBP) -Tennesine 3 Fusion Protein

A plasmid capable of producing a fusion protein of MBP (maltose binding protein) -Tennessin 3 by inserting the Tennessine 3 gene prepared in Example 2 into the vectors pMAL-p2 and pMAL-c2 having the genes of maltose binding protein. pMP-35 and pMC-35 were prepared (see Figure 3). Among them, Escherichia coli JM109 cells containing pMC-35 plasmids were grown overnight, and 10 ml of them were added to 1 L of LB medium and grown at 37 ° C for 3 hours. The IPTG was added to a final concentration of 0.3mM and shaken for 2 hours to incubate. After incubation, cells were harvested by centrifugation at 5000 rpm for 20 minutes at 4 ° C, and dissolved in 200 mM of buffer solution (10 mM Tris-Cl, pH 7.4, 20 mM NaCl). It was ultrasonically broken using Ultrasonics W-385, left on ice, centrifuged at 10000 rpm for 30 minutes at 4 ° C, the supernatant was taken, and diluted by adding 5 times the buffer solution for the column. Of these, 50ul was electrophoresed on a 15% SDS-PAGE gel by Laemmli's method to confirm the expression of the fusion protein.

In order to separate and purify the fusion protein, the amylose resin was well filled in the column, washed with the column buffer solution in an amount corresponding to 8 times the volume of the column, and the ultrasonic crushing solution obtained above was injected at an appropriate flow rate. After the sample was injected, the column was washed with 8 times the volume of the buffer solution for the column, and the column buffer containing 10 mM maltose was divided into fractions while desorbing the fusion protein bound to the resin. Each fraction was irradiated with an SDS-PAGE gel to collect only the fractions with the desired fusion protein. It was concentrated to 2 ml and 1 ul of factor Xa was added to 1 ml of this concentrate and left overnight at room temperature to allow cleavage of MBP and Tennessine 3.

Example 4

Preparation of New Recombinant DNA for Mass Expression in Escherichia Coli

Recombinant DNA for mass expression of intact Tennessine 3 in Escherichia coli was prepared by the following method (see FIG. 4).

The vector used pET-3a, an E. coli expression vector, and the insert is a coding sequence of Tennessine 3, which is a cDNA obtained from brown cell total RNA and is a PCR reaction at a binding temperature of 48 ° C. Got through. The 5'- and 3'-PCR primers used were as follows.

The product obtained through the PCR reaction had an NdeI-BamHI linker, which was cleaved with Ndel-BamHI and cloned into pET-3a to prepare a recombinant plasmid pAFT-1 (see FIG. 4).

On the other hand, Tennessine 3 has a signal peptide, which is cut off to become a mature protein. Since E. coli cells use a signal peptide to transport proteins produced by E. coli in a periplasmic space, If tenesine 3 signaling peptides also act on bacteria, they will be able to easily purify the complete form of tenesine 3. To this end, a new 5 ′ primer was prepared and cDNA containing a signal peptide was obtained by PCR, and then cloned into a pET-3a vector, which was then referred to as pAFT-2 (see FIG. 4).

In addition, in order to isolate and purify the His-taq fusion protein of Tennessine 3 using Ni ²⁺ affinity column chromatography, the Tennessine 3 cDNA was cloned into the pRSET B vector, and His-taq was the N-terminus of Tennessine 3. To prepare a plasmid pRSAF-1 capable of producing a fusion protein fused to.

[Example 5]

High Purity Purification of Tennessine 3 Expressed in Escherichia Coli

MBP-Tennesine 3 fusion protein itself is low in activity, it is difficult to obtain a high concentration of Tennessine 3 protein by using it cut to Xa, pRSAF-1 prepared in Example 4 was used.

Bacterial culture

Ampicillin was added to 10 ml of sterile LB medium to make a final concentration of 1 mM, and then inoculated by taking 2-3 colonies of Escherichia coli with pRSAF-1, and then precultured at 37 ° C. overnight. When the absorbance at 600 nm was measured at an OD value of about 0.6-1, the above solution was mixed in 1 L of LB / amp medium having the same composition as in the pre-culture, and then incubated at 37 ° C. for about 3-4 hours. When the absorbance was about 1 at 600 nm, 1 ml of 1M IPTG was added and incubated at 37 ° C. for 3 hours. Thereafter, the cultured strains were collected by centrifugation at 8,000 g for 10 minutes at 4 ° C., suspended in a small amount of 1 × binding buffer, and then subjected to power and melting at about −20 ° C. three times. Thereafter, care was taken to prevent heat and bubbles on the ice, and the cells were ultrasonically disrupted intermittently until the sample became slightly black. After centrifugation for 20 minutes at about 20,900g at 4 ℃, the supernatant was carefully taken and injected into the next step His-tag affinity column.

Isolation of Recombinant Tennessine 3 Derivatives Through His-tag Affinity Columns

The column was filled by a method of freely falling by carefully suspending about 2 ml of resin to prevent air bubbles from entering and washing the resin with about 5 times the volume of secondary distilled water. At this time, the flow rate was about 1 drop (17ul) per 4 seconds, and the flow rate was maintained at the next process. Thereafter, Ni was added to the 1X buffer (charge buffer) to charge the resin, equilibrated with the binding buffer again, and the supernatant obtained above was injected. Equilibrate again by adding 1 × binding buffer, then add 10% and 16% elution buffers in equilibrium until equilibrium, and finally add 100% elution buffer to effluent all that was bound to the column (column The silver was eluted with 6 times volume of strip buffer for reuse and stored at 4 ° C.) In order to remove excess salts and excess amidazole from the aliquots, Ultra filtration was performed using a membrane of 1000. The process of filtration, concentration, and dilution with an appropriate amount of secondary distilled water were repeated several times.

Isolation of Recombinant Tennessine 3 Derivatives by HPLC

The sample separated by the His-tag column was dried using Speed-Vac at room temperature, dissolved in a small amount of purified water for HPLC, and injected into a TSK-gel 3000SW column. At this time, the flow rate was measured at 0.3ml / min, UV absorbance at 220nm, the solvent was used a 25% CH ₃ CN solution containing 0.05% TFA. Through this process, the eluate was separated for each peak separated by SDS-PAGE, and the purity and molecular weight were determined, and the antibacterial activity was tested for each.

Example 6

Preparation of Recombinant DNA to Produce Mutant Tennessine 3 Proteins

Preparation of Plasmids for Deletion of His-tag Tennessine 3

(1) Preparation of plasmid for N-terminal deletion

The SacI / BamHI restriction site of the pAFT-2 vector containing the cDNA portion of Tennessine 3 was cleaved and treated with a klenow fragment to fill the bases, and then linked to the si to remove the SacI site. The plasmid was obtained and named "pAFT-22". The BamHI, HindIII fragment of the plasmid obtained by cloning the EcoRI, HindIII cross section of pAFT-22 to the pRSET B vector was cloned back into pGEM-3zf (+) from which the SacI site was removed and named "pJLAF5d".

(2) N-terminal deletion

By cutting the SacI restriction enzyme site of pJLAF5d vector to make 3 'overhang resistant to exonuclease, 5' overhang with BalII restriction enzyme, and then using the gene clean method Purification using The obtained DNA fragment was treated with Exo III exonuclease and fractionated over time. Each fraction was treated with SI nuclease to remove single stranded portions, and then heated to 70 ° C. to inactivate nucleases and undergo blunt-end ligation to mutant recombination of N-terminal deletions. DNA was obtained (see Figure 5).

(3) C-terminal deletion

3 'overhang was made by cleaving the SphI restriction enzyme site of the pRSAF3d (= pRSAF-1) vector, 5' overhang was made with Sa1I restriction enzyme, and the serialized vector was purified by jinclean method. As in the N-terminal deletion experiment, the obtained DNA fragment was fractionated over time by treatment with Exo III exonuclease. Each fraction was treated with S1 nuclease to remove the single stranded portion, then heated to 70 ° C. to inactivate the nucleases and subjected to blunt-ended ligation to obtain mutant recombinant DNA of the C-terminal deletion (see: 5).

Expression and isolation of deleted recombinant protein

(1) Preparation of strain for synthesizing deleted recombinant protein

For plasmids lacking the C terminus, the plasmids used in the deletion experiments did not need to be cloned again because the plasmids were capable of expressing the recombinant protein directly. Transfer to pRSET vector for expression. Since the deletion point was randomly determined, three parts of the deleted Tennessine were cloned to express the His-tag in consideration of the codon during translation. All plasmids were then introduced into E. coli BL21 (DE3) strains with T7 RNA polymerase gene for expression.

(2) Expression of deleted recombinant protein

For mass production of deleted recombinant proteins, each of BL21 (DE3) of single colonies with each plasmid was first incubated in liquid medium for about 12 hours, then diluted to 1/100 in 500 ml LB culture and incubated for another 3 hours. . When the OD ₆₀₀ value was about 1.3, IPTG was added to a final concentration of about 1 mM and further incubated for about 3 hours.

(3) Isolation and Purification of Deleted Recombinant Proteins

After protein production, the cells were harvested by centrifugation, and then frozen rapidly and resuspended in 10 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl pH 7.9) and sonicated. Subsequently, the supernatant obtained by centrifugation at 21,000xg was added to an affinity column filled with a resin of His bond bound to Ni ²⁺ , and the binding buffer and the elution buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl) were added. Only His-tag fused Tennessine 3 was eluted by a concentration gradient column chromatography method of pH 7.9). At this time, the experiment was performed using an automated liquid column chromatography device for more convenient and accurate concentration gradient column chromatography. The eluted proteins were expressed by SDS-PAGE, and then concentrated using a dialysis tube (MWCO 1000) to remove salts and lyophilization.

Preparation of Plasmids for Site-Specific Mutation Reactions

In situ mutagenesis was introduced for p5212 having the smallest DNA fragment of Tennessine 3 among the recombinant proteins obtained in the N-terminal determination experiment. For site mutations, BamHI, HindIII fragments of pHTΔN45 were cloned into the pALTER-1 vector and named "pSelHTΔN45". The colon obtained by cleaving pSelHTΔN45 with PstI restriction enzyme to treat the cleno fragment and recombination to remove the PstI site was named “pSe1HTΔN45Δ”.

(1) Design of site mutations and primers

The primers were designed to divide the amino acids of pHTΔN45 into six blocks and to block mutate each block with four alanines. At this time, a new PstI restriction enzyme site was created in the alanine block in the degenerate codon to facilitate the acquisition of the mutant codon.

(2) site specific mutation

Escherichia coli containing pSelHTΔN45Δ was infected with auxiliary phage R405 to obtain single-stranded DNA, followed by Amp repair oligomer and each mutated primer, and treated with T4 DNA polymerase to obtain single-stranded DNA. Both strands were made into a template, and then nicked by treatment with ligase. After the reaction, DNA was introduced into Escherichia coli BMH 71-18mutS, a strain having a lack of repair function, and the transformant was cultured in a medium solution containing ampicillin to obtain mutated DNA (see FIG. 6).

Example 7

Investigation of Antifungal Activity of Tennessine 3, MBP-Tennesine 3 Fusion Protein, Deletion Recombinant Protein, and Site-Specific Mutant Deletion Recombinant Protein Extracted from Brown Peel

1. Antifungal Activity of Tennessine 3 Extracted from Brown Rice Wine

(1) Culture conditions of various fungi

The media and culture conditions of the fungi used in this experiment are shown in the table below.

TABLE 1

(2) Measurement of growth of various fungi

The yeast type was diluted by diluting the liquid culture solution, and the viable counting was counted, and the filamentous type was observed under the microscope to observe the condensation of the conidia.

(3) antimicrobial activity against various fungi of crude Tenecin 3

The antimicrobial effect of c-tenesin 3 on three yeast and four filamentous fungi was investigated. Filamentous fungi and Trimorphomyces papilionaceus showed no lethal effect among the seven fungi investigated (see Figs. 7a, 7b, 8 and 9). Yeast-type fungi of Saccharomyces cerevisiae and Candida albicans were almost completely inhibited in growth at the concentration of c-Tennesine 3 at 5 mg / ml (Figs. 7a and 7b), with a slight lethal effect. These yeast fungi showed a decrease in the number of cells at concentrations of 5 mg or more when c-tenesin 3 was treated in culture. However, no lethal effects of c-Tenesin 3 were observed when they were cultured in a condition where growth did not occur, i.e., sodium citrate buffer solution (0.05 M, pH 6.5) (see FIG. 10). Thus, the lethal effect of c-Tennesine 3 was found to act only on at least growing cells.

(4) Growth and Morphological Abnormalities of Various Fungi by c-Tennesine 3

As seen previously, c-Tennesine 3 did not have a lethal effect on various fungi and mainly suppressed normal growth. Candida albicans and Saccharomyces cerevisiae treated with c-tenesine were larger than untreated strains and changed from egg-shaped to spherical. The filamentous fungi had slow germination of the spores and decreased germination rate, and mycelial growth of germinated spores was inhibited or progressed abnormally.

2. Antifungal Activity of MBP-Tennesine 3 Fusion Protein

To investigate the antimicrobial activity of MBP-Tennesine trifusion protein, single colonies of Candida albicans in SD (Sabouraud Dextrose) agar were incubated for 2 hours in SD broth. Cultured 10 ³ cells were shaken in 400 ul of 2x SD broth with MBP-Tennesine 3 fusion protein or the same volume of distilled water (control), and then a predetermined amount was taken at regular intervals and plated onto SD agar. The number was calculated and used as the stock of cell growth. As a result, about 200 ug of MBP-Tenesine trifusion protein inhibited the growth of Candida albicans (see Fig. 11) and Cryptococcus neoformans (see Fig. 12) pathogenic fungi, but growth of Aspergillus nidulans and Saccharomyces cerevisiae No significant inhibition (cf. FIG. 13).

Since the antimicrobial activity of the fusion protein may be related to MPB, the antifungal activity against Candida was examined after cutting proteins obtained from pMAL-c2 and pMC-35 with Xa (see Fig. 14). As shown in FIG. 14, both the protein obtained from pMAL-c2 and the protein cut to Xa inhibited the growth of Candida to a relatively low level, but when the MBP-Tennesine 3 fusion protein was cut to Xa, it showed significant growth inhibition. It was clear that the antifungal effect of the fusion protein was due to the Tennessine 3 protein rather than MBP. The lack of activity of the fusion protein is thought to be due to the fusion with the large protein MBP, especially the C-terminal portion that appears to be conserved compared to the antifungal protein isolated from other insects, resulting in loss of activity. It is assumed that this is because. When about 50 ug of fusion protein was cut by Xa, the Tennessine 3 concentration was estimated to be about 7 ug, at which 10 ³ cells of Canadida showed little growth (see Fig. 14).

3. Antifungal Activity of Depleted Recombinant Protein

Purified deletion proteins were tested for antifungal activity against C. albicans. The amount of proteins treated in the activity test was calculated by calculating the expected molecular weight of the deleted proteins so that the amount of water, such as 50 ug of undeleted Tennessine 3, could be treated. In anticipation of the experimental results, it was predicted that the protein lacking the important site would not have antifungal activity, but the results of the actual activity showed that all of the deleted proteins had antifungal activity unexpectedly (cf. 15). Degree). In particular, HT △ C49 and HT △ N45 in the case of the N- and C-terminal half, respectively, but did not share with each other, even though they showed the same antifungal activity. Interpreting these results, Tennessine 3 has two domains for antifungal activity, which are located at the N and C ends, respectively.

4. Investigation of Antifungal Activity of Recombinant Protein in Definite Site Mutation

Purified alanine mutant proteins were tested for antifungal activity against fungal C. albicans. The amount of proteins treated in the activity experiment was tested using the OD counting method by calculating the expected molecular weight of the deleted proteins so that an amount of moles such as 20 ug of undeleted Tennessine 3 could be processed. Experimental results show that the GHQG portion of the c-block, which shows a large similarity between about eight amino acid moieties in the 49th amino acid of Tennessine 3 and anti-fungal proteins of the Tennessine 3 family, is an important amino acid that affects antifungal activity. (Cf. Fig. 16).

As described and demonstrated in detail above, according to the present invention, Tennessine 3, a novel antifungal protein isolated from brown larvae, and its variants are produced in large quantities by genetic engineering methods. This mass-produced Tennessine 3 and its variants can be used in pharmaceutical compositions of antifungal agents with pharmaceutically acceptable ingredients.

Claims (12)

Antifungal protein (Tenecin 3) having the following amino acid sequence or functionally equivalent amino acid sequence isolated from the larva of T. molitor:

Gene of the antifungal protein having the following sequence encoding the antifungal protein (Tenecin 3) of claim 1 and its functional equivalents;

An expression vector comprising the antifungal protein (Tenecin 3) gene of claim 2. The expression vector according to claim 3, which is a protein expressible plasmid. The expression vector according to claim 3, wherein a maltose binding protein is used as a fusion partner. 4. The expression vector of claim 3, wherein the histidine is a vector capable of producing a labeled antifungal protein (Tenecin 3). Method for producing a recombinant antifungal protein (Tenecin 3) comprising the step of inducing the expression of the antifungal protein (Tenecin 3) by culturing the host microorganism transformed with the expression vector of claim 3. The method according to claim 7, wherein the host microorganism is Escherichia coli. The antifungal protein (Tenecin 3) of claim 1 wherein the signal peptide is deleted and the amino acid sequence of the mature antifungal protein (mature Tenecin 3) remaining partially deleted or substituted. The method according to claim 9, wherein amino acids 94-96, amino acids 70-96, amino acids 61-96, amino acids 48-96, 19-19 based on the amino acid sequence of the antifungal protein (Tenecin 3) comprising a signal peptide A variant characterized in that one amino acid group selected from the group consisting of amino acids 28, 19-35, 19-47, 19-55 and 19-63 is deleted. The method according to claim 9, wherein amino acids 67-70, amino acids 72-75, amino acids 77-80, amino acids 82-85, 86-86 based on the amino acid sequence of the antifungal protein (Tenecin 3) including a signal peptide A variant characterized in that one amino acid group selected from the group consisting of amino acids 89 and 91-94 is substituted with alanine. A recombinant variant comprising the step of inserting the gene encoding the variant of claim 9 into a protein expressible vector, introducing it into a host microorganism, culturing the transformant to induce the expression of the variant protein, and obtaining the same. Method of producing protein.

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