KR20040045961A - Method for mass-producing of a recombinant human tyrosinase in E. coli - Google Patents

Abstract

The present invention relates to a method for mass production of recombinant human tyrosinase protein in Escherichia coli, and more specifically, (a) amplifying the human tyrosinase gene by performing PCR using a primer pair for amplifying the human tyrosinase gene; (b) inserting the amplified human tyrosinase gene into a pET-26b (+) vector to prepare a recombinant expression vector pET-hTyr; (c) transforming Escherichia coli with the recombinant expression vector pET-hTyr; And (d) culturing the transformed Escherichia coli to express the recombinant human tyrosinase protein, and then collecting and purifying the recombinant human tyrosinase protein. The method according to the present invention has the advantage of stably expressing recombinant human tyrosinase having intact enzymatic activity without the formation of insoluble aggregates in Escherichia coli and mass production.

Description

Method for mass-producing of a recombinant human tyrosinase in E. coli

The present invention relates to a method for mass production of recombinant human tyrosinase protein in Escherichia coli, and a method for mass production of recombinant human tyrosinase protein in Escherichia coli using a recombinant expression vector pET-hTyr for expressing recombinant human tyrosinase protein.

Tyrosinase (tyrosinase, monophenol monooxygenase, EC 1.14.18.1) is a metalloprotein containing a pair of copper ions in its active site (Beltramini et al ., Biochem Bophys. Acta . 1040: 365-372, 1990) . Tyrosinase and tyrosine hydroxylase activity catalyze the hydroxylation reaction of converting monohydroxyphenol to o-dihydroxyphenol as shown in Scheme 1 below. It has a dopa oxidase activity that catalyzes the reaction of phenol to o-quinone (Mason, HS, Annu Rev. Biochem . 34: 105-184, 1965).

Tyrosinase is distributed in almost all species, including animals, plants and bacteria (Lerch, K., PNAS . 75: 3605-3609, 1978). To date, numerous tyrosinase has been isolated and purified from various kinds of organisms, and among them, research on the structure and function of tyrosinase isolated from mushrooms and mice is the most active (Mayer, AM, Phytochem. 26: 11-20, 1987). ). Plant tyrosinase is involved in browning, which may cause unnecessary browning during the processing of some fruits or vegetables (Mayer, AM, supra ). Animal tyrosinase is also involved in melanin biosynthesis (Lerner AB et al. , Physiol. Rev. 30: 91-126, 1950). Animals tyrosinase is acting on tyrosine (tyrosine, hydroxy phenylalanine) kind of amino acid in the melanin production early in-dihydroxy-phenylalanine; promote the formation of (d ihydr o xy p henyl a lanine DOPA), and further dihydroxy phenylalanine (DOPA ) To form quinone (DOPA Quinone) (Hearing, VJ et al ., Pigment Cell Res . 2: 75-85, 1989; Hearing, VJ et al ., FASEB J. 5: 2902 -2909, 1991). Melanin is produced through a series of steps from the dopaquinone thus produced. In addition, tyrosinase performs a third catalysis in the terminal portion of melanin biosynthesis as well as the two steps. That is, 5,6-di-hydroxy-indole (5,6-d i h ydroxy i ndole, DHI) catalyzes the reaction of oxidizing the indole-5,6-quinone (indole-5,6-quinone) ( Pawelek , JM, Pigment Cell Res . 4: 53-62, 1991; Tripathi et al., J. Biological. Chemistry. 267: 23707-23712, 1992). Mammalian tyrosinase is contained in melanocytes (melanocytes) distributed in the skin, hair follicles, eyes and the like, and serves to protect against damage caused by ultraviolet rays.

Human tyrosinase is a 66 kDa glycoprotein bound to the membrane of melanosomes (Bause et al., Biochem. J. 209: 331-336, 1982), skin cancers and skin pigments, including melanoma It is used for medical research of skin diseases such as deficiency. In US Pat. No. 4,898,814, cDNA encoding human tyrosinase was found and its nucleotide sequence was identified. PCT application WO 90/12869 also discloses a method for producing active human tyrosinase using non-melanocytic eukaryotic cells. In the PCT application, a recombinant expression vector is prepared by inserting DNA encoding human tyrosinase into an expression vector such as a retrovirus, and introducing the recombinant expression vector into eukaryotic cells such as fibroblasts. A technique for producing human tyrosinase by culturing eukaryotic cells has been proposed. However, the method is disadvantageous in that it is technically complicated and difficult to commercialize by mass production. To date, considerable advances have been made in understanding the molecular mechanisms that regulate gene expression of human tyrosinase, but the biochemical properties, structures, and functions of human tyrosinase are difficult to secure.

On the other hand, in the past, useful proteins such as medical proteins and industrial enzymes have been obtained in a small amount from the natural state, but the development of recombinant DNA technology has made it possible to mass-produce the useful proteins. Escherichia coli is the most widely used host cell for mass production of these useful proteins. Many recombinant useful proteins have been successfully produced by Escherichia coli, ranging from hormones such as insulin and beta-endorphin to immunomodulators such as interferon and interleukin. In addition, various substrate supply methods have been developed to cultivate a high concentration of recombinant E. coli through fed-batch cultivation, thereby enabling mass production of desired proteins with high efficiency. However, many recombinant proteins do not fully fold in Escherichia coli and form insoluble inclusion bodies that are inactivated by various mechanisms (Gongquin et al ., Arch Biochem. Biophys . 236). : 135-142). In particular, recombinant tyrosinase has been reported to form insoluble aggregates when expressed in E. coli (Kong et al ., CBP , 125: 563-569, 2000). Such insoluble protein can be easily obtained in the initial stage of separation to obtain pure protein of high purity, but does not have activity as a protein. Therefore, there is a need for denaturation and refolding processes to convert insoluble proteins into biologically active water soluble proteins. However, the process does not only require a lot of time and money, but also large amounts of protein are lost in the process. Therefore, there is an urgent need for a method for mass production of recombinant tyrosinase in water-soluble form having intact enzymatic activity in Escherichia coli.

The present inventors have previously applied for a method for producing recombinant human tyrosinase using E. coli (Korean Patent Application No. 2001-57379). The inventors of the present invention have been repeatedly researched to produce a stable mass production of recombinant human tyrosinase having high inactivation in Escherichia coli, and the method according to the present invention can produce a recombinant human tyrosinase having a higher inactivation than the method disclosed in the patent application. The present invention has been completed by elucidating that it can.

Therefore, the technical problem to be achieved by the present invention is to provide a method for mass production of a stable expression of recombinant tyrosinase in E. coli.

1 is a schematic diagram illustrating a process of inserting the human tyrosinase gene amplified by PCR into the cloning vector pCR 2.1-TOPO.

Figure 2 is a schematic diagram showing the manufacturing process of the recombinant expression vector pET-hTyr according to the present invention.

Figure 3 is a gel photograph showing the results of performing 1% agarose gel electrophoresis to identify fragments of human tyrosinase gene amplified by PCR.

Lane M: λ DNA / Hin dIII molecular weight marker

Lane 1: amplified human tyrosinase gene

4 is a gel photograph showing the results of 1% electrophoresis by treating Nde I and Xho I to the vector to identify human tyrosinase genes inserted into the pCR 2.1-TOPO-hTyr vector.

Lane M: λ DNA / Hin dIII molecular weight marker

Lane 1: human tyrosinase gene

Lane 2: pCR 2.1-TOPO-hTyr vector without restriction enzyme treatment

Lane 3: pCR 2.1-TOPO-hTyr vector treated with restriction enzyme

5 is a gel photograph showing the results of 1% electrophoresis by treating Nde I and Xho I to the vector to identify human tyrosinase gene inserted into the recombinant expression vector pET-hTyr according to the present invention.

Lane M: lambda / Hin dIII molecular weight marker

Lane 1: human tyrosinase gene

Lane 2: pET-hTyr vector treated with Nde I alone

Lane 3: pET-hTyr vector treated with Xho I alone

Lane 4: pET-hTyr vector treated with both Nde I and Xho I

Figure 6 is a gel photograph showing the results of performing 12.5% SDS-PAGE to confirm the recombinant human tyrosinase purified from the crude extract.

Lane M: molecular weight marker

Lane 1: crude extract

Lane 2: recombinant human tyrosinase purified by DEAE-Sephacel chromatography

Lane 3: Recombinant human tyrosinase by DEAE-Sephacel chromatography and His bind resin chromatography

In order to achieve the above object, the present invention

(a) amplifying the human tyrosinase gene by performing PCR using a primer pair for amplifying the human tyrosinase gene having the nucleotide sequence set forth in SEQ ID NO: 1;

(b) inserting the amplified human tyrosinase gene into pET-26b (+) to prepare a recombinant expression vector pET-hTyr;

(c) transforming Escherichia coli with the recombinant expression vector pET-hTyr; And

(d) culturing the transformed Escherichia coli to express the recombinant human tyrosinase protein, and then collecting and purifying the recombinant human tyrosinase protein.

"HTyr gene" in the present invention means a one tyrosinase (Tyr osinase) derived from the human body (h uman) gene.

Hereinafter, the present invention will be described in detail.

The present invention is characterized in that it provides a method for stably expressing human tyrosinase in E. coli so as to have intact enzymatic activity in mass production.

In one embodiment of the present invention, the human tyrosinase gene was amplified by PCR. As the tyrosinase gene that can be used in the present invention, any tyrosinase gene derived from the human body may be used without limitation, and more preferably, the human tyrosinase gene disclosed in GenBank Accession No. NM_000372 is used. The base sequence of the human tyrosinase gene used in the present invention is described as SEQ ID NO: 1. The inventors produced primer pairs for amplifying the human tyrosinase gene. The primer may be designed with reference to the nucleotide sequence of the human tyrosinase gene as set forth in SEQ ID NO: 1, and more preferably, it is designed to include a restriction enzyme site for cloning an appropriate sequence in the nucleotide sequence of the primer. Specifically, the inventors in each sequence of the primerNdeI andXhoThe restriction enzyme recognition site of I was constructed (see Table 1). Produced by the inventor The base sequences of the primer pairs for amplifying human tyrosinase genes were described as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. Subsequently, the human tyrosinase gene (hereinafter, 'hTyrGene ') was amplified (see FIG. 3).

In another embodiment of the present invention, a mass expression system for expressing the hTyr gene in E. coli was constructed. To this end, the inventors inserted the amplified hTyr gene into pET-26b (+), which is an E. coli expression vector, to prepare a recombinant expression vector pET-rTyr. First, the amplified hTyr gene was inserted into the cloning vector (see FIG. 1). In this case, the cloning vector may be used without limitation as long as it is a cloning vector for E. coli commonly used in the art, and more preferably, pCR 2.1-TOPO is used. Thereafter, to again remove the hTyr gene inserted into pCR 2.1-TOPO vector and inserts it into the pET-26b (+) vector to prepare a recombinant expression vector pET-hTyr (Fig. 2). The present invention expression of the hTyr gene vectors It is characteristic that pET-26b (+) was used.

In another embodiment of the present invention, after transforming Escherichia coli using the recombinant expression vector pET-hTyr according to the present invention, the transformed Escherichia coli was cultured to express recombinant human tyrosinase. Expression of recombinant human tyrosinase is preferably cultured by adding IPTG to a final concentration of 0.4 mM when the OD ₆₀₀ value of the culture medium is 0.3-0.4. Tyrosinase was found to be expressed in the highest amount when cultured in the above conditions (result not shown). The amount of tyrosinase activity and expression according to incubation time (4, 6, 8, 12, 24 hours) after IPTG administration were investigated. As a result, the activity and expression of tyrosinase continued to increase until 12 hours. However, the enzyme activity and the amount of expression decreased rapidly during 24 hours of cultivation, and the result was about 50% of the amount of enzyme activity and the amount of expression during the 12 hours of cultivation (result not shown). Therefore, the optimal expression condition of the recombinant tyrosinase gene according to the present invention is to incubate for 12 hours at 37 ℃ by adding IPTG to a final concentration of 0.4mM when the OD ₆₀₀ value is 0.3-0.4 in LB medium.

In another embodiment of the present invention, recombinant human tyrosinase was isolated and purified from E. coli culture. According to the present invention, recombinant human tyrosinase expressed in E. coli transformed with the pET-hTyr recombinant vector is expressed as a fusion protein in the form of 6 histidine molecules. Therefore, as a separation and purification method, it is preferable to use anion exchange chromatography, and then use affinity chromatography that is specifically adsorbed with histidine again. Specifically, in the present invention, after primary purification using DEAE-Sephacel anion exchange chromatography, it is finally obtained by using His Bind Resin chromatography. Purified. His molecules are adsorbed onto Zn ²⁺ or Ni ²⁺ through the His bind resin chromatography to purify only pure recombinant human tyrosinase.

The enzymatic properties of recombinant human tyrosinase isolated and purified according to the method of the present invention are as follows.

1) Molecular Weight: 66kDa

2) inactivity

Inactivity to tyrosine hydroxylase activity: 88.0 μmol / mg / h

Inactivity to dopa oxidase activity: 699 units / mg

3) Km value for substrate

L-tyrosine: 2.34 μM

L-Dopa: 0.31 mM

4) Inhibitory effect ( IC ₅₀ value)

Arbutin: 4.30 mM

L-ascorbic acid: 5.26 × 10 ^-3 mM

Glutathione: 2.31 × 10 ^-2 mM

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Amplification of the Human Tyrosinase Gene

1-1) Preparation of primer for amplification of human tyrosinase gene

A primer pair for amplifying a human tyrosinase gene was prepared by referring to the nucleotide sequence of the tyrosinase gene disclosed in Gene Bank Registration No. NM_000372. For easy cloning, as shown in Table 1 below, the restriction sites of Nde I and Xho I were constructed in the sequence of the primer. The base sequences of the prepared two primers were described as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. At this time, each primer was synthesized by requesting by JL Science (Seoul, Korea).

Primer pair used for amplification of human tyrosinase gene Base sequence Primer 1 5'-GGAATTC CATATG CACTTCCCTAGAGCCTGTGTCTCCTCT-3 ' Nde Ⅰ Primer 2 5'-ATCCG CTCGAG CGGTAAATGGCTCTGATACAAGCTGTG-3 ' Xho Ⅰ

1-2) Amplification, Isolation, and Purification of the Human Tyrosinase Gene

PCR was performed using two primers prepared in Example 1-1 using a human 5'-stretch plus cDNA library (Clontech, Palo Alto CA, USA) as a template. It was. PCR consisted of a total volume containing 100 ng of template DNA, 0.8 μM primer 1 and primer 2, dNTP mixture (TaKaRa) at a final concentration of 0.2 mM, 5 μl of 10 × Z-Taq buffer, and 1 μl of Z-Taq polymerase (TaKaRa). It was performed using 50 μl of PCR reaction mixture. PCR reaction was 5 seconds at 98 ℃; 10 seconds at 53 ° C; And 35 cycles of 30 seconds at 72 ℃ in one cycle. Thereafter, 1% agarose gel electrophoresis was performed to confirm the amplified PCR product, and the results are shown in FIG. 3. DNA was extracted from the gel using a Geneclean kit (BIO 101).

<Example 2>

Construction of the Recombinant Expression Vector pET-hTyr

First, the PCR product obtained in Example 1 was reacted with pCR 2.1-TOPO cloning vector (Invitrogen, Carlsbad, CA, USA) for 5 minutes at room temperature to prepare a cloning vector. The cloning vector was named 'pCR 2.1-TOPO-hTyr'. Thereafter, heat shock was applied at 42 ° C. for 40 seconds to introduce the pCR 2.1-TOPO-hTyr into E. coli (TOP10 one shot® supercompetent cell). Then, transformed Escherichia coli was selected by culturing on plate L-Broth medium containing 50 µg / ml kanamycin. One of the cultured colonies was selected and again incubated in liquid L-broth medium, after which DNA was extracted. The extracted DNA was digested with Nde I and Xho I, followed by 1% agalog gel electrophoresis. The results are shown in FIG. Then, the human tyrosinase gene was purified from the gel using a QIAquick Gel Extraction Kit (Qiagen).

The purified tyrosinase gene and pET26b (+) vector (Novagen) were reacted for 24 hours at 16 ° C. using a T4 DNA ligase. The recombinant expression vector produced as a result of ligation was named 'pET-hTyr'. Thereafter, thermal shock (42 ° C.) was added to introduce the pET-hTyr into XL1-Blue (Stratagene, USA). Transformed XL-1 blue was incubated for 12 hours in LB-agar plates containing 50 μg / ml kanamycin. One of the cultured colonies was picked up and again incubated on liquid L-broth. Plasmid DNA (pET-hTyr) was isolated from the culture using a DNA purification kit (Wizard® Plus SV Minipreps DNA Purification System, Promega). Thereafter, the DNA was digested with Nde I and Xho I, followed by 1% agarose gel electrophoresis. As a result, as shown in FIG. 5, one band corresponding to the size of the pCR 2.1-TOPO cloning vector (5360bp) and one corresponding to the size of the human tyrosinase gene (1600bp) in the pET-hTyr digested with restriction enzymes. The band of each appeared was confirmed. Thereafter, the recombinant expression vector pET-hTyr, which was confirmed to have inserted human tyrosinase using thermal shock (42 ° C), was introduced into BL21 (DE3) (Novagen). Transformed BL21 (DE3) was incubated for 12 hours in LB-agar plates added with 50 μg / ml kanamycin. Thereafter, one of the colonies grown on each plate was selected and used for mass expression of recombinant human tyrosinase.

<Example 3>

Mass Expression of Recombinant Human Tyrosinase in Escherichia Coli

BL21 (DE3) transformed with pET-hTyr was incubated at 37 ° C. for 2-3 hours in LB medium supplemented with 50 μg / ml of kanamycin. OD ₆₀₀ (optical density) values were measured at regular intervals while incubating at 37 ° C. When the OD ₆₀₀ value was about 0.3 to 0.4, the expression inducing agent IPTG (Sigma) was added so as to have a final concentration of 0.4 mM. After addition of IPTG, the culture was further performed for about 12 hours to induce mass expression of recombinant human tyrosinase. After the incubation was completed, the culture solution was collected at 4 ° C., 10,000 g for 10 minutes using a freezing centrifuge and stored at -20 ° C. for freezing.

<Example 4>

Purification of Recombinant Human Tyrosinase

The mass expressed cells of recombinant human tyrosinase were washed 2-3 times with 10 ml of 20 mM potassium phosphate buffer solution (pH 6.8; hereinafter referred to as 'Buffer A') containing 1% Triton X-100, and then the cells were washed again. It was homogenized with 10 ml of buffer solution A. Then, using an ultrasonic crusher (Sonics & Materials INC.) At 20 ℃ 30 ~ 40 watts, the amplifier (pulse) for 9 seconds under the condition of 8% of the amplitude (repeating for 1 second repeatedly for 20 minutes The bacteria were destroyed. After bacterial disruption, protein and other cell debris were separated by centrifugation at 20,000g for 20 minutes at 4 ° C. This gave a crude extract from which other cellular impurities were removed. The supernatant was adsorbed on DEAE-Sephacel ion exchange chromatography (Pharmacia Biotech, Uppsala, Sweden) equilibrated with buffer A and eluted at a rate of 1 ml / min. The eluted filtrate was collected and dialyzed three times for 8 hours with 20 mM Tris-Cl (Cl-Cl) buffer containing 1% Triton X-100 and 500 mM NaCl (pH 7.9; hereinafter referred to as Buffer B). . Subsequently, the dialysate was adsorbed on His Bind Resin chromatography (Nisagen), equilibrated with buffer B. Then, it was sufficiently washed with buffer B containing 10 mM imidazol to remove unnecessary protein. At this time, the washing is performed₂₈₀OD by measuring the value₂₈₀Wash until = 0. Thereafter, the solution was eluted at a rate of 1 ml / min with buffer B containing 100 mM imidazole. The eluate was dialyzed again with buffer A three times for 8 hours. Recombinant human tyrosinase was isolated by centrifuging the dialysate at 20,000 g for 20 minutes at 4 ° C. Purity of purified recombinant human tyrosinase was confirmed by 12.5% SDS-PAGE. As a result, as shown in Figure 6, the molecular weight of the recombinant human tyrosinase according to the present invention was found to be 66kDa. In general, mammalian tyrosinase is known to increase molecular weight due to glycosylation during post-translational modification (Imokawa and Mishima,J. Invest. Derm.85: 165-168, 1985; Imokawa,J. Invest. Derm.95: 39-49, 1990), the molecular weight of tyrosinase from which glycosylation occurred was reported to be about 70 kDa (Laskin and Paccinini,J. Biol. Chem.261: 16626-16635, 1986). However, it was confirmed from the results of FIG. 6 that recombinant human tyrosinase expressed in E. coli was not glycosylated. In addition, protein quantification was performed by performing a BSA assay (Prerce Chemical Co, Rockford IL). After reacting with BSA (bovine serum albumin) as a standard protein for 30 minutes at 37 ℃, OD₅₆₂Absorbance was measured at, and a standard curve was prepared and quantified. The results are shown in Table 3 below.

Example 5

Characterization of Purified Human Tyrosinase

5-1) Tyrosine hydroxylase activity measurement

Tyrosine hydroxylase activity was measured according to Husain et al . (Husain et al ., J. Invest. Dermatol . 78: 243-252, 1982). After performing a hydroxylation reaction with the concentration of L-dopa in the range of 0 to 30 mM, a standard curve for L-dopa was prepared. The standard curve was used to determine the amount of L-wave generated after the reaction. In the reaction solution, add 50 μM potassium phosphate buffer (pH 6.8) to 100 μM L-tyrosine, L-dopa of each concentration, 4 mM ascorbic acid and 100 μl of human tyrosinase purified in Example 4, and total 2.5 ml. Was prepared. Thereafter, the reaction was carried out at 37 ° C. for 3 hours. At this time, the control was allowed to react without adding purified human tyrosinase. Thereafter, according to the method of Ram et al . (Ram et al ., J. Biol. Chem . 267: 23707-23712, 1992) by using fluorescence (excitation) at 360nm, the emission (emission) at 490nm The intensity at this time was measured. The activity of the enzyme was measured by converting the measured value to the concentration of L-dopa. The specific activity of tyrosine hydroxylase activity of the purified human tyrosinase according to the present invention was determined as the number of moles of L-dopa (mmol) relative to the amount of enzyme (μg) per hour (h). The results are shown in Table 2 below.

Tyrosine Hydroxylase Activity of Human Tyrosinase According to the Invention Purification stage Specific activity (μmol / mg / h) Crude extract 1.8 ± 0.05 DEAE-Sephacel Chromatography 11.0 ± 0.12 His bind resin chromatography 88.0 ± 1.50

As shown in Table 2 above, it was confirmed that the activity of tyrosine hydroxylase activity of the recombinant human tyrosinase finally purified by His bind resin chromatography was increased by about 88 times compared to the crude extract. In addition, the activity of tyrosinase purified by tyrosinase in human HeLa cells was compared to that of 22.3 ± 0.01 pmol / μg / h (Spritz et al ., J. Invest. Dermato. , 109: 207-212). , 1997), it can be seen that recombinant human tyrosinase expressed in E. coli has better tyrosine hydroxylase activity according to the present invention.

5-2) Dopa oxidase activity measurement

Activity measurement of dopa oxidase was performed according to the method of Fling et al . (Fling et al . J. Biol. Chem . 238: 2045-2053, 1963). It was measured at 475 nm that L-dopa, a substrate of dopa oxidase, was converted to the browning product of dopachrome by tyrosinase. Dopachrome has a maximum absorbance value with a maximum concentration at 475 nm, and the molar extinction coefficient at this time is 3600 (M ^-1 cm ^-1 ). 3 mM L-dopa was added to 50 mM Tris-HCl buffer solution (pH7.5) and allowed to stand at 37 ° C. for 10 minutes, and then 30 μl of purified human tyrosinase in Example 4 was added thereto, followed by 3 minutes at 475 nm. The change in absorbance was measured. At this time, the unit of activity of the enzyme was determined as 1 unit (μmole / min) to generate 1 μmole of dopachrome per minute. In addition, the specific activity was 1 unit per 1 mg of enzyme. The results are shown in Table 3 below in comparison with recombinant human tyrosinase (Korean Patent Application No. 2001-57379) produced in Escherichia coli using the pHis-Tyrosinase expression vector prepared in the inventor's previous study.

Dopa Oxidase Activity of Recombinant Human Tyrosinase Purification stage Total activity Total protein (mg) Inactive (units / mg) yield(%) Fold Recombinant human tyrosinase according to the present invention Crude extract 5700 400 14.3 100 One DEAE-Sephacel Chromatography 4460 64 69.7 78 5 His bind resin chromatography 1922 2.75 699 34 49 Recombinant Human Tyrosinase of Patent Application No. 2001-57379 Crude extract 136 350 0.4 100 1.0 DEAE-Sephacel Chromatography 64 46 1.4 47 3.5 His bind resin chromatography 26 One 26.0 19 65.0

As shown in Table 3, the amount of protein of the recombinant human tyrosinase produced according to the method of the present invention (the amount of protein of crude extract) and the amount of protein of the recombinant human tyrosinase produced according to the method disclosed in Patent Application No. 2001-57379 It can be confirmed that almost similar. However, the total activity of recombinant human tyrosinase according to the present invention was significantly higher than the total activity of recombinant human tyrosinase of Patent Application No. 2001-57379, and thus it was confirmed that the inactivation increased about 36 times. This is a result showing that the recombinant human tyrosinase produced in Escherichia coli using the pET-hTyr vector according to the method of the present invention was expressed as an enzyme having intact activity without forming insoluble aggregates. In addition, it was confirmed that the amount of the finally purified enzyme was significantly higher in the present invention. This indicates that the method according to the invention can produce recombinant human tyrosinase with high inactivation in E. coli in high yield.

5-3) Investigate the effect of substrate concentration

5-3-1) Effect on the Concentration of L-Tyrosine

The effect of substrate concentration on the recombinant human tyrosinase according to the present invention was investigated with the concentration of L-tyrosine, which is a substrate, in the range of 0.01-1 mM. It was carried out according to the same method as the activity measurement method of tyrosine hydroxylase of Example 5-1). The Km value of the recombinant human tyrosinase according to the present invention was calculated by Lineweaver et al . (Lineweaver et al ., J. Am. Chem. Soc. , 56: 658-666, 1934). As a result, the Km value for L-tyrosine of the recombinant human tyrosinase according to the present invention was calculated to be 2.34 μM. This K m value (0.2mM) of the tyrosinase extracted from the previous study the Km value of the human tyrosinase prepared in the inventors (0.17mM) (Republic of Korea Patent Application No. 2001-57379 call) and melanoma cells (melanoma cell) ( Sangjin et al ., Korean Biochem. J. 26: 632-637, 1993). From this, it can be seen that the affinity of the recombinant human tyrosinase according to the invention for the substrate L-tyrosine is at least 100 times higher. The results also show that the higher order structures of recombinant human tyrosinase according to the present invention are fully folded.

5-3-2) Effect on the concentration of L-dopa

The influence of the substrate concentration on the recombinant human tyrosinase according to the present invention was investigated with the concentration of another substrate, L-dopa, in the range of 0.05-5 mM. The waveguide oxidase activity measurement method of Example 5-2) was performed according to the same method. Then, Km value of the recombinant human tyrosinase according to the present invention for L-dopa was calculated according to the same method as 5-3-1). As a result, the Km value for L-dopa of the recombinant human tyrosinase according to the present invention was calculated to be 0.31 mM. This was almost similar to the Km value (0.4 mM) for L-dopa of purified tyrosinase in melanoma cells (Kang et al ., Korean Biochem. J. , 26: 632-637, 1993). From this, it can be seen that there is almost no difference in the binding force to L-dopa in the recombinant human tyrosinase and melanoma cells expressed in E. coli according to the present invention.

5-4) Determination of Inhibitory Effect

In order to measure the inhibitory effect of the inhibitor (inhibitor) on recombinant human tyrosinase according to the present invention, the inhibitory effect was measured by using arbutin, L-ascorbic acid, glutathione as an inhibitor. Each inhibitor was prepared so that the final concentration was 0.03 to 3 mM, respectively. After adding the inhibitor of each concentration and the recombinant human tyrosinase according to the present invention, the dopa oxidase activity was measured to measure the remaining activity. At this time, L-dopa was used as a substrate. The concentration of inhibitor ( IC ₅₀ ) at 50% inhibition of enzyme activity was determined from the plot for each concentration. The results are shown in Table 4 below.

IC ₅₀ values for each inhibitor Inhibitor IC ₅₀ (mM) Arbutin 4.30 ± 0.17 L-ascorbic acid 5.26 × 10 ^-3 ± 0.18 Glutathione 2.31 × 10 ^-2 ± 0.09

As described in Table 4 above, the IC ₅₀ value for arbutin was found to be 4.30 mM. This is about 75% lower concentration of tyrosinase extracted from melanoma cells compared to the IC ₅₀ value (18.9 mM) for arbutin. From this it can be seen that arbutin shows a better inhibitory effect on the recombinant human tyrosinase according to the present invention. In addition, the IC ₅₀ value of glutathione was 2.31 × 10 ⁻² mM, showing the lowest concentration. This is consistent with studies showing that sulfur-containing substances such as glutathione exhibit strong tyrosinase activity by forming complexes through coordinating bonds with tyrosinase copper ions (Wood and Schallreuter, Biochim Biophys Acta. 1074: 378-385, 1991). .

As described above, in the present invention, recombinant human tyrosinase was stably expressed in E. coli and mass produced. The method according to the present invention has the advantage of stably expressing recombinant human tyrosinase having intact activity without the formation of insoluble aggregates in Escherichia coli, thereby producing a large amount. In addition, since recombinant human tyrosinase produced in large quantities in E. coli has intact enzymatic activity, it can be usefully used for medical research of skin diseases such as skin cancer and skin pigment deficiency.

Claims (5)

(a) amplifying the human tyrosinase gene by performing PCR using a primer pair for amplifying the human tyrosinase gene; (b) inserting the amplified human tyrosinase gene into a pET-26b (+) vector to prepare a recombinant expression vector pET-hTyr; (c) transforming Escherichia coli with the recombinant expression vector pET-hTyr; And (d) culturing the transformed Escherichia coli, expressing the recombinant human tyrosinase protein, and then collecting and purifying the recombinant human tyrosinase protein. The method of claim 1, wherein the human tyrosinase gene of step (a) has a nucleotide sequence as set forth in SEQ ID NO: 1. The method of claim 1, wherein the primer pair of step (a) is characterized in that it has a nucleotide sequence described in SEQ ID NO: 2 and SEQ ID NO: 3. The method of claim 1, wherein the purification of the step (d) is performed by DEAE-Sephacel anion exchange chromatography and His Bind Resin chromatography in sequence. How to. Recombinant human tyrosinase protein prepared according to the method of claim 1.