KR100585269B1 - Halobacterium salinarum Thioredoxin having anti-oxidation activity isolated from Halobacterium salinarum - Google Patents

Abstract

The present invention relates to thioredoxin, which has an antioxidant activity isolated from Halobacterium salinarum, a basophilic bacterium that grows under not only high salt but also strong UV conditions of 25% or more.

According to the present invention, it was confirmed that thioredoxin of the present invention has excellent antioxidant activity, protects DNA nicking from metal catalyzed oxidation stress, and recovers about 20% of oxidative damage of skin caused by reactive oxygen species. The protein could be used as a biopharmaceutical as well as in cosmetics to reduce oxidative damage to the skin.

Description

Thioredoxin having anti-oxidation activity isolated from Halobacterium salinarum}

1 is a photograph showing the amplification product of the Trx gene sequence from H. salinarum .

Figure 2a is a cleavage map of the pET-28a vector (Novagen) which is an expression vector used in the present invention.

2B is a cleavage map of the recombinant vector pET-28a: trx of the present invention.

Figure 3 shows the protein sequence analysis through the Trx nucleotide sequence of H. salinarum .

4 shows E. coli , Human, and H. This is a comparison of Trx multiple sequence alignment of salinarum .

5 is a photograph showing the H. salinarum Trx his-tag mixed protein induced by the addition of 1 mM IPTG to E. coli BL21 (DE3).

6 is a purified SDS-PAGE photograph of recombinant protein of Trx isolated from H. salinarum expressed in E. coli.

7 shows ESI-Q TOF MS / MS spectrum and MASCOT search results of the protein of the present invention.

8 is a graph comparing the Trx activity of human Trx and H. salinarum by the Insulin reduction assay.

Figure 9 shows the growth curves of three E. coli transformed T. H. salinarum to a live E. coli, a Trx loss mutation and a Trx loss mutation.

10 shows the results of antioxidant activity measurement through in vivo hydrogen peroxide tolerance bioassay.

11 is a result of measuring the DNA damage protection effect by metal-catalyzed oxidation (MCO) metal ions in vitro.

Figure 12a is a graph comparing the death of cells treated with Trx of H. salinarum according to the hydrogen peroxide concentration, and Figure 12b shows the death of cells according to the concentration of Trx of H. salinarum at 1 mM hydrogen peroxide concentration It is a graph comparing.

The present invention relates to thioredoxin, an antioxidant protein present in halobacterium salinarum , a basal microorganism that survives salt concentration and oxidative stress, and its use in antioxidant, DNA damage protection and skin oxidation damage prevention.

Some microorganisms exist in extreme environments of temperature, pressure, salinity, and pH without specific ranges. The microorganism, called extremophile, has properties that can be useful in industrial processes because it produces biocatalysts that are active in extreme environments. Among extremophile, basophilic microorganisms live in balance with the outside by accumulating salt in cells (KCl <4 M, NaCl> 5 M). Recently, there has been increasing interest in the interaction of proteins in high salt concentrations, and sequencing of H. salinarum , a type of basophilic microorganism, is unknown, indicating the possibility of protein identification of basophilic microorganisms.

The thioredoxin considered would be the effect of antioxidant enzymes useful in the search for the genes of H. salinarum to select a protein of unknown H. salinarum were selected. H. salinarum is a microorganism that survives oxidative stresses such as ultraviolet rays and there has been no evaluation of the antioxidant effect of thioredoxin on this microorganism.

Thioredoxin (Trx) is a small protein of about 12 kDa and is present in both eukaryotes and prokaryotes. The active protein portion of Trx is CGPC-. In addition, Trx has many biological activities, such as (a) Trx acts as a growth factor, (b) removes hydrogen peroxide that is toxic in cells, and (c) Trx is a ribonucleotide reductase in bacteria. It plays a role in binding DNA to important factors related to transcription and transcriptional activity, and (d) affects the activity of nuclear transcription factor kB (NF-kB), which is a transcription-related factor in eukaryotic cells. Affects tumors and breaks disulfide bonds of other oxidized proteins, helping to reactivate. Because of this biological activity, Trx has recently been of interest as an anti-cancer drug as well as an antioxidant protein drug that protects cells from damage.

Therefore, the present inventors have identified the Trx present in H. salinarum , a basophilic microorganism that withstands high salt concentration and oxidative stress, and confirmed that this protein has excellent antioxidant effect, DNA damage prevention effect, and skin oxidation damage prevention effect. The present invention has been completed.

Therefore, the main object of the present invention is to provide a thioredoxin protein having excellent antioxidant activity.

Another object of the present invention to provide an antioxidant, DNA damage protection agent, and skin oxidation damage prevention cosmetic composition using the thioredoxin.

In order to achieve the object of the present invention, the present invention provides a thiorredoxin having an antioxidant activity having the amino acid sequence of SEQ ID NO: 2 isolated from Halobacterium salinarum .

Halobacterium salinarum is a basophilic bacterium that grows not only at 25% high salt but also under strong UV light conditions, and its enzymes are likely to maintain their activity under extreme conditions. In the present invention, thioredoxin having an antioxidant effect is isolated and purified from the whole protein of H. salinarum that is strong under ultraviolet light, and then applied to the E. coli system for over-expression. Purified. Thereafter, the purified thioredoxin protein was characterized and found to have excellent antioxidant activity.

In order to achieve another object of the present invention, the present invention provides a recombinant vector pET-28a: trx having a cleavage map of Figure 2b inserted with a thioredoxin gene comprising the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the thioredoxin gene was cloned and sequenced by an automatic sequencer, and the nucleotide sequence thereof was represented by SEQ ID NO: 1. At this time, the recombinant vector cloned into the expression vector is pET-28a: trx.

In order to achieve another object of the present invention, the present invention provides E. coli cells transformed with the recombinant vector of the present invention.

In the present invention, the recombinant vector containing the thioredoxin gene, which has an antioxidant effect, in the whole protein of H. salinarum , which is strong under ultraviolet light conditions, is applied to the E. coli system and over-expressed, and then yields a high yield. The protein was purified.

In order to achieve another object of the present invention, the present invention provides an antioxidant containing the thioredoxin of the present invention as an active ingredient.

In the present invention, in order to test the effect as an antioxidant of the present invention, it was confirmed through the insulin reduction assay that the Trx of the present invention (H. salinarum) has a better reducing power than Trx of human, and through the in vivo hydrogen peroxide tolerance bioassay It was confirmed that Trx of the present invention has excellent antioxidant activity.

In order to achieve another object of the present invention, the present invention provides a DNA damage protection agent containing the thioredoxin of the present invention as an active ingredient.

In the present invention, in order to test the effect of the DNA damage protector of the present invention, the Trx of the present invention through the DNA damage protection measurement by metal-catalyzed oxidation (MCO) metal ions in vitro has excellent DNA damage protection effect Confirmed.

In order to achieve another object of the present invention, the present invention provides a cosmetic composition for preventing skin oxidative damage comprising the thioredoxin of the present invention as an active ingredient.

In the present invention, in order to test the effect as a cosmetic for preventing skin oxidation damage of the present invention, after treatment with reactive oxygen species in human skin cells and measured the death by oxidative stress as a result of the skin oxidation damage excellent Trx of the present invention It was confirmed that there was an prevention effect.

Antioxidants and DNA damage protection agents of the present invention can be prepared by methods known in the pharmaceutical art in the form of pharmaceuticals or health foods, powders, granules by themselves or mixed with pharmaceutically acceptable carriers, excipients, diluents, etc. It may be prepared and used in the form of a tablet, capsule, or injection. They can also be administered orally or parenterally.

The dosage for administering thioredoxin according to the present invention as an active ingredient may be appropriately selected according to the age, sex, condition, and symptoms of the patient's need, preferably 0.1-10 mg of tea per day for adults. Oredoxin may be administered.

The cosmetic composition of the present invention is characterized by containing the thioredoxin protein in an amount of 0.001 to 50% (w / v) based on the whole composition. If the content is lower than 0.001%, it is difficult to expect the substantial antioxidant and skin protection effects of the present invention, and if the content is higher than 50%, it is difficult to maintain the properties of the cosmetic composition and affect the stability of the product.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions in addition to thioredoxin protein as an active ingredient, and include, for example, conventional solvents, stabilizers, solubilizers, emulsifiers, vitamins, pigments and perfumes. Adjuvants, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, but preferably, lotion, nutrition lotion, nutrition cream, massage cream, nutrition essence, pack, makeup base, foundation, body oil , Hair oil, shampoo, rinse is characterized in that it is prepared in a formulation selected from the group consisting of. Irrespective of the formulation of the cosmetic composition, as long as it contains the thioredoxin protein of the present invention, it has the antioxidant effect and skin protection effect of the present invention, eliminates free radicals generated in the skin, and has an intracellular antioxidant system protection effect. It can exhibit the effect of preventing and delaying skin aging caused by oxidation by the action of the back.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Cell Culture

H. salinarum (ATCC 33171), a bacteriostatic bacterium living in extreme environments, was grown at 35 ° C and 180 rpm in a medium containing 1 g / L K2HPO4, 10 g / L yeast extract and 4.3 M NaCl (pH 7.2). . Cells were harvested by centrifugation at 8000 rpm for 10 minutes in the mid-exponential phase.

HaCaT cells, human non-blast cells, were distributed from N. E Fusenig (German Cancer Reaserch Center, Heidelberg, Germany), DMEM media and 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 mg / ml The cells were incubated in streptomycin-treated medium and contained 37% CO2 and 5% carbon dioxide.

Mutant E. coli strains lacking Trx were requested from Wisconsin University (Madison, WI, USA, http://www.genome.wisc.edu/sequencing.htm ) of the E. coli genome project centrer. Obtained.

Example 2 Genomic DNA Preparation and PCR Amplification of the trx Gene

Genomic DNA according to the manufacturer's instructions using a commercially available G-spin genomic DNA extraction kit (iNtRON Biotechnology, Seoul, Korea) when H. salinarum growing in 4.3 M salts has a value from OD 600 to 1.0 Was extracted.

Similar to the strain of the present invention and previously sequenced H. sp . Based on the Trx of NRC-1, each primer containing the restriction enzyme HindIII was constructed in the forward EcoR1 / reverse: 5'-CCC GAATTC ATGACTGTCACCCTGAAAGAC-3 '(forward primer) and 5'-AAA AAGCTT TCAGGCGCCCGCGGACTCGAT- 3 '(reverse primer). PCR reaction was carried out with 17.5 ng genomic DNA, 10X reaction buffer, 2.5 uM MgCl 2, 100 uM dNTP, 2.5 U Taq polymerase and the total volume was 50 ul. PCR conditions were 95 ° C for 1 minute 40 seconds (once), 95 ° C for 1 minute, 58 ° C for 50 seconds, 72 ° C for 1 minute (30 repetitions), and 72 ° C for 10 minutes (once). Was used. PCR products were identified by 1% agarose gel. As a result, it was confirmed that the Trx gene of 300 bp length was amplified as shown in FIG. 1. 1 is a photograph showing the amplification product of the Trx gene sequence from H. salinarum .

Example 4 Cloning into Expression Vector

The identified PCR product was finally elutioned using a QIA prep spin miniprep PCR purification kit, and the restriction enzyme reaction of the elutioned Trx gene and the expression vector pET-28a vector (Fig. 2A, Novagen, Madison, WI) with EcoR1 and HindIII After the reaction at 37 ° C. for 1 hour, the recombinant Trx and the pET-28a vector were conjugated by T4 DNA ligase to prepare a recombinant vector pET-28a: trx (FIG. 2B). The prepared recombinant vector was transformed into E. coli BL21 (DE3) using heat-shock.

Example 5 DNA sequencing

The resulting white colonies were confirmed by colony PCR for the presence of insertion sequences. Colonies containing complete insertion sequences were taken and incubated in 5 ml LB with 10 mg / ml kanamycin. Plasmids were extracted using the QIAprep Spin Miniprep kit (Qiagen), and the cloned genes were then automatically sequenced by ABI 3700 sequencer (Applied Biosystems, CA, USA). The resulting gene nucleotide sequence is shown in SEQ ID NO: 1. In addition, the sequence of the protein encoded by the base sequence was analyzed through the base sequence. The amino acid sequence of the protein is shown by SEQ ID NO. Through the above protein analysis, it was confirmed that CGPC-, a protein sequence showing Trx activity, appeared. Figure 3 shows the protein sequence analysis through the Trx nucleotide sequence of H. salinarum . In addition, the similarity and similarity between TrX of E. coli and TrX of human being, which TrX was isolated and purified commercially, was confirmed by multiple sequence alignment. The agreement between Trx of E. coli and TrX of H. salinarum was 36%, and the similarity was 63.1%. The TrX of human and TrX of H. salinarum showed 39.7% and 53.8% similarity. This is because the position of CGPC amino acid as well as the position of Trypsine in the front and Lysine in the back matched, which showed high agreement and similarity. 4 shows E. coli , Human, and H. This is a comparison of Trx multiple sequence alignment of salinarum .

Example 6 Expression and Purification of Recombinant H. salinarum Trx in E. coli System

In order to overexpress and purify the transformed H. salinarum Trx gene, transformed E. coli BL21 (DE3) was inoculated in LB media containing 10 mg / ml of kanamycin at 1%, and the final concentration was about 0.7 at OD value. Phosphorus 1 mM of IPTG was treated, and then incubated at 25 ° C. for 4 hours, and the degree of overexpression was confirmed by SDS-PAGE. Thereafter, purification was performed using 50% Ni-NTA resin.

H. salinarum Trx is encoded in the pET-28a vector, and when it is expressed as a protein, the hig-tag protein of the H. salinarum Trx back base sequence is also expressed. The expression of H. salinarum Trx transformed into E. coli was confirmed by the method used by Smith and Johnson. As shown in FIG. 5, when 1 mM of IPTG was added and E. coli was incubated at 25C, protein expression increased rapidly, particularly after 4 hours. 5 is a photograph showing the H. salinarum Trx his-tag mixed protein induced by the addition of 1 mM IPTG to E. coli BL21 (DE3). Each pellet incubated for 0, 1, 2, 4 and 6 hours was confirmed by SDS-PAGE, and the expression of recombinant protein was observed when 1 mM of IPTG was added at 4 hours. The magnitude of the expression of the protein is predicted to be about 12 kDa. Hig-tagging H. salinarum Trx mixed protein can be obtained easily using Ni-NTA resin under conditions that do not denature the protein. Ni-NTA resins can be easily purified by binding to His-tag, the amino acid behind the expressed protein while competing with His-tag. 6 is a purified SDS-PAGE photograph of recombinant protein of Trx isolated from H. salinarum expressed in E. coli. The size of recombinant protein containing Trx of H. sallinarum in E. coli was 12 kDa, indicating that the purity of elution 2 and 3 was high during purification. The yield of recombinant protein in T. H. sallinarum in E. coli was summarized. The total amount of recombinant protein grown in 20 ml LB broth and the amount of protein in each step were measured. The protein content of Elution 1 was 25%, and the protein content of Elution 2 was 13%.

Table 1 Purification Yield Table of Recombinant Proteins

Example 7 ESI-Q TOF MS / MS Analysis of Proteins

ESI-Q TOF MS / MS analysis was performed to confirm that the purified protein was indeed Trx of H. salianrum. First, destain the cut SDS-PAGE gel spot with destaining solution, add Acetonitrile and dehydrate until the gel turns white. The dried gel pieces were incubated for 45 minutes on ice with 20 μl of 50 mM ammonium bicarbonate containing 0.2 μg of trypsin (promega). After removing the reaction solution, 30 μl of 50 mM ammonium bicarbonate was added and reacted overnight at 37 ° C. for in-gel trysin digestion. The peptide solution is desalted using a C18 nano column (home made). MS / MS is performed with nano-ESI on a Q-TOF2 mass spectrometer (Micromass, Mancheser, UK). To identify proteins, all MS / MS spectra were examined for protein sequences in the NCBInr database using the MASCOT search program (www.matrixscience.com).

The purified protein was confirmed by SDS-PAGE, and the peptide was separated by trypsin, and the result of ESI-Q TOF MS / MS analysis showed a peak as shown in FIG. 7. 7 shows ESI-Q TOF MS / MS spectrum and MASCOT search results of the protein of the present invention. Purification 1, 2, and 3 were confirmed whether the protein purified by ESI-Q TOF was Trx from H. salinarum using a single band in the SDS-PAGE gel. Mascot search showed that the purified protein was Trx of H. salinarum and its coverage was found to be 71% consistent.

Example 8 Determination of enzyme activity through reducing power of insulin

Holmgren, A. (1979, J Biol Chem 254 (19) 9627-9632) Method to detect the antioxidant activity of Trx by Holmgren, A. (1979, J Biol Chem 254 (19) 9627-9632) Insulin disulfide reduction assay was performed. 600 ul of reaction mixture contained 0.1M potassium phosphate, 2 mM EDTA, 0.13 mM insulin. Human Trx was used as positive control and negative control was used as DTT without Trx. Assay was initiated with the addition of 0.3 mM DTT and the absorbance was measured at 650 nm at 1 minute intervals on a spectrophotometer .

Trx and H of Human. the activity of Trx salinarum were compared through the insulin reduction assay to determine the ability to break the disulfide bond of the insulin -chain. Human Trx showed a rapid response after 10 minutes but decreased activity after 30 minutes. However, Trx of H. salinarum showed activity after 15 minutes, higher activity than human Trx, and remained active after 30 minutes. 8 is a graph comparing the Trx activity of human Trx and H. salinarum by the Insulin reduction assay.

Example 9 In vivo assay for hydrogen-peroxide in E. coli system

To test the susceptibility of E. coli cells to H 2 O 2, mutant E. coli from which the Trx gene was deleted was measured for resistance to hydrogen peroxide. Experiments were performed using mutant (Mut) E. coli and H. salinarum Trx with Trx removed (Mut + ArcTrx) E. coli, and wild type E. coli, the control of the experiment. As a result of comparing the growth curve of the type of bacteria, it was confirmed that the growth curve did not affect the growth of bacteria even if the Trx of the external gene, H. salinarum, was added. To determine whether Trx of H. salinarum inhibited the growth of E. coli, three types of E. coli transformed Trx of H. salinarum to wild type E. coli, mutants that lost Trx and mutants that lost Trx. Growth curves were compared. The antioxidant effects of three E. coli were measured on LB plates containing hydrogen peroxide. As a result, there was no difference in the growth curves of all E. coli, and E. coli, which transformed Trx of H. salinarum , grew 5.3 times more than the mutant in LB plate treated with 0.5 mM hydrogen peroxide. Figure 9 shows the growth curves of three E. coli transformed T. H. salinarum to a live E. coli, a Trx loss mutation and a Trx loss mutation.

In addition, when 0.5 mM and 0.75 mM hydrogen peroxide were included in the LB medium, it was measured how the three types of bacteria were affected in growth. Each bacterial strain was cultured in LB medium, and when the OD value was 0.5 or more, the final volume of 1 mM IPTG was added and the protein was expressed. After dilution so that the value at OD 600 was 0.2. Diluted by 2/2 LB plate, LB plate containing 0.5 mM hydrogen peroxide, LB plate containing 0.75 mM hydrogen peroxide was spotted by 37 ℃, 14 hours incubation, and observed the presence of colonies. LB plate is a positive control, LB plate containing 0.75 mM hydrogen peroxide is a negative control. We should pay attention to ArcTrx (Trx of H. salinarum) rather than Mut in LB plate containing 0.5 mM hydrogen peroxide. genes in the mutants) had higher growth colonies. There was a significant difference in dilution from 1/4 to 1/16. Therefore, it was observed that Trx, which is not present in Mut, plays an important role in oxidative stress such as hydrogen peroxide. 10 shows the results of antioxidant activity measurement through in vivo hydrogen peroxide tolerance bioassay.

Example 10 DNA Damage Protection Effect Test by MCO (metal-catalyzed oxidation) Assay

The enzymatic effect of DNA damage under metal-catalyzed oxidation (MCO) stress was tested. The reagent for MCO reaction was 20 μM FeCl 3, 4 mM DTT, Trx of the purified H. salinarum at a concentration of 25 ug / ml to 150ug / ml, the total amount was adjusted to 50 ul with distilled water, and then 2 hours at 37 ℃. After that, 800 ng of pUC18 DNA was added and reacted for 2 hours and 30 minutes, which was confirmed using 1% agarose gel. In this experiment, metal ions damage the DNA by changing its supercoil form to a linear form. H. salinarum's Trx was determined to protect against DNA damage caused by metal ions by varying the H. salinarum's Trx concentration from 25 ug / ml to 150 ug / ml. As a result, it was confirmed that there is a protective effect against DNA breaks against metal ions when more than 100ug / ml. 11 is a result of measuring the DNA damage protection effect by metal-catalyzed oxidation (MCO) metal ions in vitro.

(Lane 1: pUC18 DNA control; lane 2: pUC18 DNA in the MCO reaction mixture; lane 3-6: pUC18 DNA in the MCO reaction mixture with various concentration of ArcTrx 150, 100, 50, and 25 g / ml; lane 7 pUC18 DNA in the MCO reaction mixture, with BSA as a control protein)

Example 11 Cytotoxicity Assay by Cytotoxicity Assay

Cytotoxicity test by oxidative stress was carried out through MTT assay. In a 96 well plate, 2X10 ⁵ HaCaT cells were grown in DMED containing 10% FBS, and after 1 day, the cells were wiped with PBS to remove the medium, and hydrogenated peroxide was treated in DMED without FBS. After time incubation, the medium was removed, treated with MTS, and then incubated for 1 hour and 30 minutes, which was then measured at 490 nm with an ELISA reader. When human skin cells are treated with reactive oxygen species, hydrogen peroxide, the cells are killed by oxidative stress. Toxicity by hydrogen peroxide was measured by MTS cell viability assay. Toxicity of H. salinarum Trx in 10 ug / ml and different concentrations of hydrogen peroxide was measured. As a result, cytotoxicity was low when H2 salinarum was 1 mM. When treated with Trx, it was found that the cell damage caused by hydrogen peroxide was reduced by 18%. Therefore, it was confirmed that Trx of H. salinarum has a protective effect against damage of hydrogen peroxide, which is an active oxygen species in the medium. However, 100 mM hydrogen peroxide itself was very toxic, so the effect of Trx of H. salinarum could not be expected. And at 1 mM hydrogen peroxide conditions. Different concentrations of Trx in salinarum were measured to protect apoptosis from oxidative stress. As a result, cell death was reduced according to the amount of the administered protein. Figure 12a is a graph comparing the death of cells treated with Trx of H. salinarum according to the hydrogen peroxide concentration, and Figure 12b shows the death of cells according to the concentration of Trx of H. salinarum at 1 mM hydrogen peroxide concentration It is a graph comparing.

As described above, according to the present invention, thioredoxin was isolated and purified from H. salinarum , which has not yet been identified. The protein has excellent antioxidant activity, protects DNA nicking from metal catalyzed oxidation stress, It has been confirmed that the oxidative damage of skin caused by about 20% is recovered, so that the protein can be used as a biopharmaceutical as well as a raw material of cosmetics that reduces oxidative damage of the skin.

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Claims (6)

Thioredoxin having antioxidant activity having the amino acid sequence of SEQ ID NO: 2 isolated from Halobacterium salinarum . A recombinant vector pET-28a: trx having a cleavage map of FIG. 2B into which a thioredoxin gene comprising the nucleotide sequence of SEQ ID NO: 1 is inserted; E. coli cells transformed with the recombinant vector of claim 2. Antioxidant containing thioredoxin according to claim 1 as an active ingredient. DNA damage protection agent containing thioredoxin according to claim 1 as an active ingredient. Cosmetic composition for preventing skin oxidative damage comprising thioredoxin according to claim 1 as an active ingredient.

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