KR20010071097A - THIF, a novel stress-regulating protein which interacts with thioredoxin - Google Patents

Abstract

PURPOSE: Provided is a novel regulatory protein, THIF, which inhibits the activity of thioredoxin and enhances cell death by binding to thioredoxin, an intracellular reducing material. The THIF can be used in regulating stress, and is thus useful to biotechnology and medical science. CONSTITUTION: A THIF(thioredoxin-interacting factor) gene has base sequence represented by sequence No. 1, No. 2, No. 3 or No. 4 and expresses THIF protein which binds to thioredoxin.

Description

THIF, a novel stress-regulating protein which interacts with thioredoxin}

The present invention relates to a novel stress regulator protein THIF and its coding gene, which has the property of binding to intracellular reducing agent thiolidoxin (TRX) and inhibiting the function of TRX and promoting sandpaper death. The present invention finds the THIF gene by the yeast two-hybrid method, clones the gene, constructs a THIF expressing recombinant plasmid, and expresses THIF under various stress conditions (hydrogen peroxide treatment, radiation and ultraviolet irradiation, high temperature shock, etc.). By investigating the effects of this, the present invention relates to clarifying the stress regulating function of THIF.

Thiolidoxin (TRX) is a 12kDa-sized protein that is primarily responsible for restoring function by reducing proteins that have been oxidized and lost their function in cells. TRX is found in most organisms such as mammals, plants and microorganisms. It has two cysteine residues in its active position, which reduces oxidized other proteins as it is oxidized, and is itself responsible for NADPH and flaoprotein TRX reductase. Is reduced again to restore activity. However, TRX has a number of biological functions in addition to reductive action. That is, it is known that hydrogen is supplied to ribonucleic acid reductase essential for DNA synthesis and secreted outside the cell to proliferate immune cells, fibroblasts and various cancer cells. It is essential for early pregnancy, inhibits HIV expression in phagocytes, and regulates intracellular transcriptional regulators such as TFIIIC, BZLF1, NF-κ, glucocorticoid receptor, and Ref-1. In addition, the role of antioxidants to reduce the active oxygen generated by hydrogen peroxide, TNF-α is widely known. Recently, it has been found to be an inhibitor of ASK-1, an enzyme involved in cell death signaling.

The present inventors believe that finding a new protein that binds to TRX, which plays a physiologically important role, is of great significance, and has studied the yeast two-hybrid method to isolate a new stress-regulating protein called THIF and its coding gene. It became. In particular, the THIF coding gene has been previously announced to increase expression by vitamin D ₃ , but its function is unknown, it was found in the gene database search results. As a result of the study test of the present inventors, it was found that the THIF protein is not only bound to TRX and inhibits TRX function but also deeply related to the cell death.

Accordingly, it is an object of the present invention to provide an amino acid sequence of a novel stress regulator protein THIF that binds to thiolidoxin (TRX) and a base sequence of its coding gene.

Hereinafter, the configuration and effect of the present invention will be described in detail.

Figure 1 shows the identification of THIF genes that bind to thiolidoxin (TRX), where A is the amino acid sequence of THIF, B is the THIF protein coding sequence, and C is the GST-Pull binding of TRX and THIF in 293 cell lines. down assay, D shows the binding of the THIF fragment (134-395 amino acid site) to TRX, E to the mutant TRX,

Figure 2 shows the expression of THIF, A is the production of transforming vector pFLAG-THIF, B is the production of pLXSNTHIF, C is the expression level of the tissues of the mice by mRNA in vitro method, D is a single cell Shows the results of analysis by using a cell fluorescence spectrometer,

Figure 3 shows the result through the enzyme reaction THIF inhibits the reduction of TRX,

Figure 4 shows that THIF gene expression is regulated by a variety of substances, A by H ₂ O ₂ or ionomycin in KMls cell line, a mouse T cell, B by TGF-β1 in primary cultured mouse lung cells , C is the result of experiments by gamma-ray radioactivity and UV light in mouse T cell KMls cell line, and D is the result of experiment by Northern analysis on the change of THIF mRNA expression by high temperature shock in mouse fibroblast NIH3T3.

Figure 5 shows that THIF competitively interferes with the binding of TRX between PAG and ASK-1, the intracellular substances involved in apoptosis,

6 is an experimental result showing that THIF is involved in cell proliferation and death, A is a control experiment showing the expression of THIF in THIF-introduced cells, B is the THIF introduction reduced the proliferation of NIH3T3 cell line by TGF-β1 As a result, C shows that the death of NIH3T3 cell line by H ₂ O ₂ is increased by the introduction of THIF,

Figure 7 shows the results of THIF on IL-6 expression and JNK activity, A is the result of measuring the increase in IL-6 expression by TGF-β1 in THIF-introduced cells by RT-PCR method, B shows the results of experiments in which JNK activity is increased by H ₂ O ₂ in cells with increased THIF expression.

In light of previous studies, TRX has been known to regulate phosphatase, such as ASK-1, which plays an important role in the redox signaling system in cells. To make it clear, a new technology called yeast two-hybrid screening has been used to isolate a novel protein, the thiredoxin-interacting factor (THIF) and its coding gene, which can bind to TRX. THIF protein isolated by the present inventors has the property of binding to thiolidoxin to regulate the reducing power of thiolidoxin and thereby regulating the stress of cells as described in detail below.

The scope of the present invention includes the amino acid sequence of the stress regulator protein THIF, and also functionally equivalent protein molecules in the form in which any amino acid residue in the amino acid sequence is substituted with another amino acid which is functionally equivalent and a protein fragment exhibiting the same or similar activity. Or even derivatives thereof. The scope of the present invention also encompasses nucleotide sequences encoding THIF proteins. Those skilled in the art will appreciate that the base sequence of the present invention also includes a base sequence by overlapping the genetic code inferred from the protein amino acid sequence.

The present invention relates to the identification of THIF, the THIF-binding protein coding gene THIF, the tissue distribution and intracellular localization of THIF, the effects of THIF on the reducing power of TRX, the expression of THIF gene, and the overexpression of THIF by TRX, PAG, and TRX. And the effect of THIF on the stress response by H ₂ O ₂ , on the binding between and ASK-1.

Experimental methods related to genetic engineering of the present invention are described in Sambrook, J. et al .: Molecular Cloning. A laboratory Manual . 2nd ed., Cold Spring Harbor Laboratory. Cold Spring Harbor.New York., 1989; Ausubel, F. et. al .: Short Protocols in Molecular Biology . 3rd.John & Wiley Sons, Inc., 1995).

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples. However, the following examples are only for illustrating the present invention in detail, and the scope of the present invention is not limited only to these examples.

Example 1 Exploration of THIF Gene

Experimental Example 1: THIF search by yeast two-hybrid screening

Bait plasmid was prepared by synthesizing the gene of TRX by reverse transcriptase polymerase chain reaction (RT-PCR) method in human keratocytes cDNA library and inserting into pEG202 plasmid. The prepared bait plasmid was introduced into the yeast into which the pYESTrp plasmid containing the cDNA library of the A20 cell line, which is a mouse B cell, was introduced, and the yeast cells that showed positive reaction on the culture plate that gave a blue color when the protein binding to TRX were expressed. Got a clone.

The cloned gene sequence was analyzed by a Gene-Bank database, which turned out to be a mouse gene corresponding to human VDUP1, which was previously discovered but its function was not revealed, and the present inventors identified a thiredoxin-interacting factor (THIF). Was named. Currently, the THIF gene is registered in the Gene-Bank under accession number AF173681. When cloned from the cDNA library, a part of 5 'side was searched in a truncated form, and the remaining part was inferred from the EST database to prepare a cDNA containing the entire gene by polymerase chain reaction. Analysis of the entire THIF gene in several databases encodes a protein consisting of 395 amino acids (FIG. 1A), which has 94% similarity to the human gene and no distinct functional site was found. The nucleotide sequence of the THIF protein coding gene is shown in Figure 1b.

THIF was able to bind strongly to TRX in yeast two-hybrid screening system. To determine whether it can bind to mammalian cells, THIF was constructed using expression vectors capable of expressing flagged THIF and GST attached TRX. Cell lines were transfected. The transduced cells were disrupted to precipitate GST-TRX with glutathione-sepharose beads, followed by Western blotting experiments with anti-flag antibodies. THIF bound to GST-TRX in the same manner as the yeast two-hybrid experiments. It was confirmed to precipitate together (Fig. 1c).

In order to test whether the N-terminally truncated THIF (134-395) can also be combined with TRX, GST-precipitation experiments were carried out in the same manner as above to confirm that it precipitated with GST-TRX. It was found that there was a TRX binding site between the residues (FIG. 1D). In addition, in order to determine whether cysteine residues 32 and 35, which are known as active sites of TRX, are required for binding to THIF, the two cysteine residues were replaced with serine residues. CS) and did not bind (Fig. 1e), it was determined that the cysteine residues 32 and 35 are required for binding to THIF.

Experimental Example 2 cDNA Cloning

(1) THIF gene cloning: The gene obtained by the yeast two-hybrid method was inserted into the pFLAG-CMV expression vector as part of the THIF gene to prepare pFLAG-THIF _(134-395) .

Synthesis of polymerase chain reaction start primers 1 and 2 having the following sequence to amplify the 5'-cutting site using the EST database to find the entire sequence of the THIF gene, and reverse transcriptase-polymerase chain from A20 B cell library As a result, the entire THIF gene was obtained and inserted into pFLAG-CMV and pLXSN plasmids to prepare pFLAG-THIF (FIG. 2A) and pLXSN-THIF (FIG. 2B). Escherichia coli E. coli DH10B / pFLAG-THIF and E.coli DH10B / pLXSN-THIF transformed with each of the vectors prepared above were assigned to KCTC 0704 BP and KCTC 0705, respectively. Deposited as BP.

Primer 1 (forward):

5'-GCCAAGCTTATGGTGATGTTCAGAAGAT-3 '

Primer 2 (reverse):

5'-TTCACTAGATCTCACTTCG-3 '

(2) TRX gene cloning: after synthesizing the starting primers 3 and 4 having the following nucleotide sequence for cloning the entire expression region of the TRX gene, and amplified using human keratinocyte cDNA library, EcoRI and BamHI restriction enzyme Was inserted into pBluescript KS (+).

Primer 3 (forward):

5'-GCCCGAATTCAAGATGGTGAAGCAGATC-3 '

Primer 4 (reverse):

5'-GGCGGATCCTTAGACTAATTCATTAATGGTG-3 '

In addition, they were inserted into pEBG and pFLAG-CMV vectors, respectively, to make GST and flagged TRX. In order to replace the two cysteine residues of the active site with serine residues to make the TRX lost activity, primers 5 and 6 having the following sequencing were synthesized to generate a specific site mutation kit (ExSite PCR-Based site-directed mutagenesis kit, Stratagene, La Jolla, CA) was used to generate mutant TRX and insert it into the pEBG vector to produce GST-TRX (CS).

Primer 5 (forward):

5'-CCACGTGGTCTGGGCCTTCCAAAATHAT-3 '

Primer 6 (reverse):

5'-ATCATTTTGGAAGGCCCAGACCACGTGG-3 '

Experimental Example 3 Analysis of THIF Gene Expression

(1) Northern blotting assay: KM1s-8 in PMA (50 nM), ionomycin (1 μg / ml), H ₂ O ₂ (200 μM), γ-ray (20 Cy), UV light in mouse T cell lines (100J / m ² ), and NIH3T3 cell lines were treated with 42 ° C. high temperature, and mouse primary cultured lung fibroblasts were treated with TGF-β1, followed by RNA with Tri-Reagent (Molecular Research Center, Cincinnati, OH). Was extracted. 30 μg of the extracted RNA was electrophoresed on an agarose gel and then adsorbed onto a nylon membrane (Gene Screen Plus, NEN Life Science Products, Boston, MA). The membrane was allowed to attach radiolabeled THIF gene fragments in 65 ° C. ExpressHyb solution (Clonetech, Palo Alto, Calif.), Followed by sufficient washing to specifically attach to THIF mRNA attached to the membrane.

(2) Assay by reverse transcriptase polymerase chain reaction: MMLV reverse transcriptase (Promega) 2.5 unit, dNTP (2.5 mM each of dATP, dCTP, dGTP and dTTP) were added from 3 μg of RNA and 37 μm of random primer DNA was added thereto. After 1 hour of reaction to synthesize complementary single-stranded DNA, a part of 91/20) was added to each specific starting primer DNA 10 pmole, 0.5 unit Taq DNA polymerase, 10X polymerase chain reaction buffer, 4 μl of dNTP. Into the reaction solution containing the back was carried out a polymerase chain reaction of 95 ℃ 10 minutes, 30 chain reactions (95 ℃ 1 minutes, 55 ℃ 1 minutes, 72 ℃ 1 minutes), 72 ℃ 10 minutes. 10 μl of the reaction product was taken and electrophoresed at 100 V for 15 minutes on a 1% agarose gel to confirm the result. Each starting primer DNA is as follows.

Primer for THIF Amplification

Primer 7 (forward)

5'-GCCGAATTCAAGATGGTGAAGCAGATC-3 '

Primer 8 (reverse)

Primer for TRX Amplification

Primer 9 (forward):

5'-GTGGTGGACTTCTCTGCTAC-3 '

Primer 10 (Reverse):

5'-GCTGGTAGCTGGTTACACTT-3 '

Primer for IL-6 Amplification

Primer 11 (forward):

5'-CTCAGCCCTGAGAAAGGAGA-3 '

Primer 12 (reverse):

5'-AAAGCTGCGCAGAATGAGAT-3 '

Primer for TGF-β1 Amplification

Primer 13 (forward):

5'-CGGGAGGCCAGCCGCGGGAC-3 '

Primer 14 (reverse):

5'-GGGTTGTTTGGTTGTAGAAAAC-3 '

Primer for β-actin amplification

Primer 15 (forward):

5'-GTGGGGCGCCCCAGGCACCA-3 '

Primer 16 (Reverse):

5'-CTCCTTAATGTCACGCACGATTTC-3 '

Experimental Example 4 Cell Culture and Transduction

HeLa, 293, NIH3T3, mouse lung fibroblasts were cultured in DMEM medium supplemented with 10% bovine serum, L-glutamine, antibiotics, and the like, and KMls cell lines were cultured in RPMI medium (including 10% bovine serum, glutamine, antibiotics). Cell lines were incubated in a 60 mm dish for transient transduction into 293 and HeLa cell lines, followed by transduction of recombinant DNA by calcium-phosphorus precipitation. PLXSN and pLXSN-THIF recombinant DNA were transduced with lipofectamine (Lipofectamine, GIBCO-BRL, Grand Island, NY) to make cell lines permanently transduced with NIH3T3 cell line, and 0.5ng / ml of G-418 Cell lines were established under conditions such that they were cultured in the added medium so that only permanent transduced cell lines could live.

Experimental Example 5: Immunochemical Analysis of Cells

Transduced HeLa cell lines were incubated on sterilized thin glass plates, washed with phosphate buffer, dried and fixed with cold methanol: acetone (1: 1) mixed solution. The immobilized cell line was reacted in phosphate buffer solution with 1% BSA for 20 minutes and mouse anti-flag M2 monoclonal antibody (Eastman Kodak Co., Rochester, NY) or control was added to 1/100 of mouse for 1 hour. After the reaction, the mixture was washed five times with phosphate buffer. The washed cell line was reacted with goat FITC-attached anti-mouse IgG antibody (Becton Dickinson, San Jose, Calif.) Diluted at 1/200, and then washed again with phosphate buffer. Finally, the cell lines were adsorbed with glycerol solution and observed with a confocal laser scanning microscope. Confocal scanning microscopy was performed using a Leica DAS upright microscope (Leica Lasertech GmnH, Heildelberg, Germany) to which Lesica TCS 4D was connected.

Experimental Example 6 Precipitation and Western Analysis of GST-Fusion Protein

24 hours after transduction, 293 cells were harvested and precipitated in phosphate buffer solution and destroyed solution (20 mM Hepes (pH 7.9), 100 mM KCl, 300 mM NaCl, 10 mM EDTA, 0.1% NP-40, Protease inhibitor mixed solution) After breaking for 1 hour at, it was reacted with glutathione-sepharose (Pharmacia) at 4 ° C. for 2 hours. Wash 5 times with the above destructive solution, boil in SDS-PAGE sample solution, electrophoresis on SDS-modified polyacrylamide gel and stain with Coomassie dye or Immuno-Blot PVDF membrane (BIO-RAD, Hercules, CA) Western blotting experiments were performed by reacting with an anti-flag M2 antibody or an anti-HA 12CA5 antibody (Boehringer Mannheim, Mannheim, Germanyy) to confirm with an ECL system (Enhanced chemoluminoscence system, American pharmacia Biotech, Piscataway, NJ). .

Experimental Example 7 JNK Activity Measurement

500 μg of cell extracts were immunoprecipitated with JNK antibody (pharminggen) and reacted with GST-c-Jun (1-79) in the presence of [γ- ³² P] dATP, followed by electrophoresis and drying on 12% polyacrylamide gel. It confirmed by photosensitive to the line film.

Experimental Example 8: Measurement of TRX Activity by Insulin Antigen Response

Temporally transduced cells were disrupted with cell disruption solution (20 mM Hepes (pH 7.9), 100 mM KCl, 300 mM NaCl, 10 mM EDTA, 0.1% NP-40, protease inhibitor containing solution), 20 μl of DTT TRX was reduced by mixing with active buffer (50 mM hepes, pH 7.6), 1 mM EDTA, 1 mg / ml BSA, 2 mM DTT, adjusting the volume to 70 μl and reacting at 37 ° C. for 20 minutes. Take 40 μl of this and mix with 200 μl of 1 M Hepes (pH 7.6) solution, 40 μl of 0.2 M EDTA, 40 μl of 40 mg / ml NADPH, 500 μl of 10 mg / ml insulin and again add 10 μl of thiolidoxin reductase (100 A412 units / ml) The reaction was carried out at 37 ° C. for 20 minutes. After the reaction, 6M guanidine-HCl (guanidine-HCl) and 1mM DTNB mixed solution was added to stop the reaction, and the absorbance was measured at 412 nm wavelength.

Experimental Example 9: Analysis of Cell Proliferation

The cells were suspended at 5 × 10 ³ cells / ml in DMEM medium supplemented with 10% bovine serum, incubated for 12 hours in 200 μl per well in a 96-well culture plate, and further cultured with H ₂ O ₂ or TGF-β1 for 24 hours. . Then 0.5 μCi of ³ H-thymidine was added to each well, followed by incubation for 6 hours, and then collected on a glass fiber filter paper using a multiple cell harvester (Inotech) to a liquid scintillation counter (Beckman). ^{The content of 3} H-thymidine was measured.

Experimental Example 10 Analysis of Apoptosis Using Flow Cytometer

pLXSN or pLXSN-THIF were permanently introduced and incubated for 12 hours in a culture plate per 24 wells at 2 × 10 ⁵ cells / ml and treated with H ₂ O ₂ . After the incubation, trypsin treatment to remove adherent cells, adding propidium iodide (PI, 5μg / ml) to measure the PI content (FL-2 channel) and cell size (forward light scatter) at the same time, the cell size Apoptosis was measured by considering the cells showing decreased and increased PI content as dead cells.

Example 2 Investigation of Expression of THIF

Experimental Example 1 Tissue Distribution and Intracellular Position of THIF

In order to determine the histological distribution of THIF, mRNA expression was measured by Northern blotting from various tissues of male Balb / c mice. THIF transcripts were expressed in tissues such as heart, lungs, thymus, spleen, testis and muscle, and relatively small amounts in brain and liver tissues. The results are shown in Figure 2C. TRX, on the other hand, was expressed in large amounts in liver tissue. A computerized sequence analysis (PSORT II, National Institute of Basic Biology, Genoment) was used to determine the intracellular location of THIF, suggesting that it was present in the cytoplasm. Temporally transduced and immunochemical analysis with anti-flag antibody confirmed that it is also present in the cytoplasm. The results are shown in FIG. 2D.

Experimental Example 2: Effect of THIF on the Reducing Power of TRX

We investigated whether THIF inhibits TRX function by binding to TRX. Intracellular TRX was reduced by thiolidoxin reductase, and the reducing power was tested whether THIF could inhibit the reducing power of TRX using the principle that sulfuric acid of oxidized insulin could be sulfuric. Overexpressing THIF in the 293 cell line, it was found that not only the reducing power of TRX present in the cells, but also the reducing power of TRX overexpressed by transducing the TRX expression vector with THIF (FIG. 3). At this time, the function of the inhibited TRX was about 50%, and it was an experiment in which THIF was an inhibitor of TRX.

Experimental Example 3 Expression Control of THIF Gene

TRX is an important factor in the redox regulation of cells, and its gene expression is known to be regulated by a variety of stresses, so first we examined whether THIF expression changes under various stresses (including oxidative stress). Treatment of mouse T cell line KMls-8 with 200 μM of H 2 O 2 significantly increased THIF transcript expression and decreased THIF expression while increasing calcium expression (ionomycin) treatment with TRX (FIG. 4A). When TGF-β1 was treated for 24 hours in primary lung fibroblasts of mouse lung, THIF expression was significantly increased while TRX expression was not changed (FIG. 4B). Radioactive gamma or ultraviolet irradiation increased THIF expression in KMls-8 cells after 5 hours (FIG. 4C). When heat treated to NIH3T3 cells, THIF expression was increased after 1 hour (FIG. 4D). Comparing the expression of TRX and THIF genes showed that THIF was much more sensitive to stress. From these results it was concluded that THIF is closely involved in the response to various stress situations.

Experimental Example 4: Effect of THIF Overexpression on the Binding Between TRX and PAG, TRX and ASK-1

Proliferation Associated Gene (PAG) is known to reduce the H ₂ O ₂ by binding to TRX and examined whether THIF inhibits TRX-PAG binding. 5A shows that overexpression of THIF reduces the binding between TRX-PAG by 50%. In addition, MAPKKK called ASK-1, which is involved in apoptosis caused by oxidative stress, is also known as an important TRX binding protein. Therefore, THIF overexpression is also TRX-ASK in an experiment that examined whether THIF is involved in TRX-ASK-1 binding. It was also found that -1 binding was also inhibited (FIG. 5B). The results suggest that THIF binds TRX competitively with PAG or ASK-1.

Experimental Example 5: H ₂ O ₂ Effect of THIF on stress response

The known physiological function of PAG is known that TRX acts as an electron donor when protecting cells from apoptosis induced by serum removal, ceramide, etoposide, and the like. In addition, inhibition of binding between TRX-ASK-1 has also been reported to result in greater damage of cells by oxidative stress, so NIH3T3 cell lines show how overexpression through transduction of THIF is affected by oxidative stress. THIF was permanently transduced. Overexpression of THIF was verified by reverse transcriptase chain reaction (FIG. 6a), and overexpression through THIF transduction did not affect cell shape or cell growth compared to the control group transduced with the vector only. However, it was found that the cell growth of THIF overexpressing group was slower than TGF-β1 treatment or serum removal in medium (FIG. 6B). In addition, 100 μM H ₂ O ₂ treatment, THIF overexpression group (79.2% ± 16.5) showed increased cell death compared to the control group (50.1% ± 14.2) (Fig. 6c).

In previous reports, free radicals have been known to be involved in the expression of inflammatory response factors such as IL-6, and it can be seen that IL-6 expression is increased when H ₂ O ₂ is treated in THIF transduced cells (FIG. 7A). ). In addition, it was reported that JNK activated by ASK-1 was affected by H ₂ O ₂ treatment, indicating that JNK activity was increased in THIF transduced cells (FIG. 7B). In conclusion, THIF expression was found to further aggravate cell damage by oxidative stress.

As described above, the THIF protein of the present invention inhibits the reducing power of thiolidoxin by binding to thiolidoxin and thus has a stress regulating property of the cell. Thus, the present invention utilizes THIF, a thiolidoxin binding and stress regulator. It can be used to control stress is a very useful invention in the biotechnology and medical industry.

Claims (10)

A THIF (thioredoxin-interacting factor) gene having a nucleotide sequence as shown in SEQ ID NO: 1 encoding a THIF protein that binds to thioredoxin. THIF (thioredoxin-interacting factor) protein having an amino acid sequence as shown in SEQ ID NO: 2 that binds to thioredoxin. Claim 1 SEQ ID NO: 3 of the gene encoding the THIF protein that binds to thiolidoxin (thioredoxin). A protein having an amino acid sequence as shown in SEQ ID NO: 4 encoded by the base sequence of claim 3. Recombinant expression vector comprising the THIF gene of claim 1. The plasmid pFLAG-THIF of claim 5, wherein the recombinant vector is contained in E. coli DH10B / pFLAG-THIF (KCTC 0704BP). The plasmid pLXSN-THIF of claim 5, wherein the recombinant vector is contained in E. coli DH10B / pLXSN-THIF (KCTC 0705BP). Microorganisms transformed with the recombinant expression vector of claim 5. The transforming microorganism is E. coli DH10B / pFLAG-THIF (KCTC 0704BP). The method of claim 8, wherein the transforming microorganism is E. coli DH10B / pLXSN-THIF (KCTC 0705BP).