KR100450307B1 - C-terminus protein of human nucleolar phosphoprotein hNopp140 and the gene thereof - Google Patents

Abstract

The present invention relates to a C-terminal region protein and its gene of the human nucleolar phosphoprotein (hereinafter, referred to as "hNopp140"), and more particularly, to specifically bind to doxorubicin. C-terminal region protein of hNopp140 and the gene encoding the same. The C-terminal region protein of hNopp140 of the present invention has a property of binding to doxorubicin, an anticancer substance in cells, and plays an important role in the mechanism of cytotoxicity or anticancer activity, and reduces side effects of doxorubicin and improves anticancer effects. It can be usefully used.

Description

C-terminus protein of human nucleolar phosphoprotein hNopp140 and the gene approximately}

The present invention relates to a C-terminal region protein and its gene of the human nucleolar phosphoprotein (hereinafter, referred to as "hNopp140"), and more particularly, to specifically bind to doxorubicin. C-terminal region protein of hNopp140 and the gene encoding the same.

Doxorubicin is an anthracycline family of antibiotics isolated from streptomysis and consists of an anthracycline ring linked to an amino sugar via a glycosidic bond. Doxorubicin causes side effects such as nausea, anorexia and alopecia, but is widely used for the treatment of various tumors and malignant leukemias (Blum and Carter, Ann. Intern. Med. , 1979, 80, 249-259). Because doxorubicin has four ring structures, it is inserted between base pairs of DNA and interferes with DNA replication or transcription (Crooke et al., Mol. Pharmacol. , 1978, 14, 290-298; Merivether and Bachur, Cancer Res . , 1972, 32, 1137-1142), and is also known to affect the activity of DNA denaturases such as DNA phase isomerase (Tewey et al., Science , 1984, 226, 466-468).

Although the DNA binding properties of doxorubicin have been shown to be important for its anticancer activity, no detailed cytotoxicity mechanisms are known, but there are reports that doxorubicin will interact with other cellular components other than DNA (Chuang and Chuang, Biochemistry). , 1979, 18, 2069-2073). Doxorubicin has a much higher binding affinity for double-stranded DNA than thermally denatured DNA, but transcription by RNA polymerase II is inhibited by doxorubicin to a similar degree for both types of DNA (Chuang and Chuang, Biochemistry , 1979, 18, 2069-2073). In addition, free radical semiquinones produced from doxorubicin (Sinha and Chignell, Chem. Biol. Interact ., 1979, 28, 301-308; Berlin and Haseltine, J. Biol. Chem ., 1981, 256, 4747-4756) and doxorubicin It is reported that interactions with components in the cell membrane cause cytotoxicity (Tritton and Yee, Science , 1982, 226, 4666-468; Hasmann et al., Biochem. Pharmacol ., 1989, 38, 305- 312). These reports suggest that there may be various intracellular components that interact with doxorubicin, and some of them may be targets for anticancer activity. Because cancer treatments are widely used in cancer patients, identifying cellular components, especially proteins, that specifically interact with doxorubicin is essential for reducing its side effects and improving anticancer effects.

Various methods have been used to find proteins that specifically act on proteins or ligands, but there are many limitations and difficulties in identifying ligands, especially proteins that work with small molecular weight chemicals. Recently, a method of allowing cDNA of human tissues to appear on the surface of phage has been used to find proteins or peptides that specifically bind to immobilized ligands (Crameri et al., Eur. J. Biochem . , 1994, 226, 53-58; Jespers et al., Gene , 1996, 173: 179-181). For example, FKBP12 could be selected from the T7 phage library for FK506 (Sche et al., Chem. Biol. , 1999, 6, 707-716). Therefore, phage display has emerged as a very suitable method for finding proteins having specific affinity with ligands having a small molecular weight.

Accordingly, the present inventors have tried to find intracellular components that can specifically bind doxorubicin and become targets for anticancer activity. As a result, the inventors discovered a C-terminal region protein of hNopp140 that specifically binds doxorubicin, The present invention has been completed by revealing the mechanism of anticancer activity, which can be useful for reducing side effects of doxorubicin and improving anticancer effects.

Accordingly, it is an object of the present invention to provide a protein of the C-terminal region of hNopp140 and its genes which can be usefully used to reduce the side effects of doxorubicin and to improve anticancer effects by revealing the mechanism of cytotoxicity or anticancer activity.

A of FIG. 1 is a graph showing the number of the phage in the eluent and the solution after washing steps, each bio-panning,

▥ = phage count in solution after each biopanning step,

▧ = phage count in the eluate of each biopanning step,

Figure 1 B is an electrophoresis picture showing the results of PCR (polymerase chain reaction) of the eluate of each biopanning step,

Figure 1 C is an electrophoresis picture showing the PCR results of the randomly extracted phage for each biopanning step,

2 is a photograph showing the results of the plaque hybridization (hybridization) from about 1000 plaques in each biopanning step,

And A of Figure 3 showing the results of SDS-PAGE (polyacrylamide gel electrophoresis) of purified proteins and protein dephosphorylation, and the phosphorylated protein electrophoresis image,

M: standard molecular weight marker, 1: Trx-hNopp140C,

2: dephosphorylated Trx-hNopp140C, 3: phosphorylated Trx-hNopp140C,

4: Trx-hNopp140C phosphorylated with ³² P-ATP,

*: Alkaline phosphatase,

And B of Figure 3 showing the results of Urea-PAGE of the purified protein, protein dephosphorylation, and the phosphorylated protein electrophoresis image,

1: Trx-hNopp140C, 2: dephosphorylated Trx-hNopp140C,

3: phosphorylated Trx-hNopp140C,

Figure 4 is a bar graph showing the results of the enzyme linked immunosorbent assay (enzyme linked immunosorbent assay) to determine the binding affinity between doxorubicin and protein,

FIG 5 A is a graph using a fluorescence emission characteristic of doxorubicin measuring the binding affinity of the protein,

Figure 5 B is a graph measuring the binding force between doxorubicin and hNopp140C using a BIAcore machine.

In order to achieve the above object, the present invention provides a protein of the C-terminal region of hNopp140 that specifically binds doxorubicin.

The present invention also provides a gene encoding the protein.

Hereinafter, the present invention will be described in detail in the above order.

First, the present invention provides a protein of the C-terminal region of hNopp140 that specifically binds doxorubicin. All C-terminal proteins of hNopp140 that specifically bind to doxorubicin are included in the scope of the present invention. In particular, the protein of the C-terminal region of hNopp140 that specifically binds to doxorubicin is preferably the protein of the C-terminal region of hNopp140 having the amino acid sequence of SEQ ID NO: 1 .

In another aspect, the present invention provides a gene encoding a protein of the C-terminal region of all hNopp140. The gene encoding the protein of the C-terminal region of hNopp140 is preferably a gene having a nucleotide sequence described in SEQ ID NO: 2 .

Hereinafter, the process of separating the C-terminal protein and its gene of hNopp140 having the property of binding to doxorubicin of the present invention, and confirming their binding to doxorubicin is as follows.

The present inventors specifically bind to immobilized ligands by binding human liver cDNA to the surface of phage to find intracellular components that can specifically bind doxorubicin and become targets for anticancer activity. Biopanning methods for finding proteins or peptides were used (Crameri et al., Eur. J. Biochem ., 1994, 226, 53-58; Jespers et al., Gene , 1996, 173: 179-181).

First, doxorubicin bound to biotin was fixed to a plate covered with streptavidin, and a phage library containing human liver cDNA was added to the doxorubicin-fixed plate to react, and then used in the biopanning step. DNA was isolated from the phages eluted in the biopanning to analyze the DNA sequence to obtain the sequence described in SEQ ID NO: 2 . This was compared with other DNA sequences of GeneBank using the BLAST program, and the sequence described in SEQ ID NO: 2 was found to be a gene encoding the C-terminal region of hNopp140. The amino acid sequence of the C-terminal portion of hNopp140 is set forth in SEQ ID NO: 1 .

The C-terminal region protein of hNopp140 of the present invention found through the biopanning process plays an important role in the mechanism of cytotoxicity or anticancer activity by binding to doxorubicin in cells, and is useful for reducing side effects of doxorubicin and improving anticancer effects. Can be used.

The present inventors amplify the cDNA insert by PCR from the T7 phage containing the gene of the C-terminal region protein of hNopp140 for the expression and purification of the h-Nopp140 C-terminal region protein found through the biopanning process. Then, the protein was cloned into a pTrx vector made by cloning a thioredoxin gene in a pET28a expression vector (Novagen) to prepare a protein in which thioredoxin and hNopp140C were combined, which was named "Trx-hNopp140C".

Reported that hNopp140 is phosphorylated to a very high degree by casein kinase II (Pai et al., J. Cell. Sci ., 1995, 108, 1911-1920; Chen and Yeh, Biochem. Biophys. Res. Commun ., 1997, 230 , 370-375) as a result of confirming that the purified Trx-hNopp140C also has the same properties of phosphorylation, the original state and the dephosphorylated Trx-hNopp140C shows the same mobility in SDS-PAGE and Urea-PAGE Phosphorylated protein was much slower on the SDS-PAGE due to the increase in molecular weight by phosphate groups than the original protein and was observed at the same position when ³² P-ATP was used for phosphorylation. In addition, the Urea-PAGE was found to move faster due to the negative charge of the increased phosphate group of the phosphorylated protein (see FIG. 3 ). The results indicate that the protein expressed in Escherichia coli maintains the original form of hNopp140.

In addition, the binding specificity of hNopp140C to doxorubicin of the present invention was confirmed by ELISA (Enzyme Linked Immunosorbent Assay). Namely), while a weak positive signal is shown, whereas for Trx-hNopp140C, a strong positive signal is shown (see FIG. 4 ). In addition, as a result of analyzing the binding force of the h-Nopp140 C-terminal doxorubicin of the present invention by the fluorescence analysis, the intensity of the emission spectrum was increased by Trx-hNopp140C, but phosphorylated Trx-hNopp140C by the phosphorylated Trx-hNopp140C No change was seen (see A of FIG. 5 ).

Biosensor analysis showed strong binding signal when Trx-hNopp140C was flowed, and dissociation constant with doxorubicin was 4.52 × 10 ^-6 M. On the other hand, if only Trx was flowing, no coupling signal could be confirmed, and thus, the coupling signal between Trx-hNopp140C was found to be due to hNopp140C. As in fluorescence analysis, phosphorylated Trx-hNopp140C showed no binding to doxorubicin (see B of FIG. 5 ).

Phosphate protein 140, the human nucleus of the present invention found through the biopanning process from the above results, plays an important role in the mechanism of cytotoxicity or anticancer activity by binding to doxorubicin in cells, and is useful for reducing side effects of doxorubicin and improving anticancer effects. Can be used.

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 Isolation of Protein Binding to Doxorubicin

<1-1> Fixation of doxorubicin

We treated the plates with streptabitin and fixed doxorubicin to biotin to fix doxorubicin. Doxorubicin and biotin are reacted at room temperature for 3 hours after mixing 8 mM doxorubicin with 2 mM sulfo-NHS-biotin in 50 mM sodium bicarbonate buffer (pH 8.0). It was. Sulfo-NHS-biotin remaining after the reaction was treated with 50 mM glycine for 1 hour to allow all reactions to occur. The mixture was diluted 20-fold in PBS (phosphate buffered saline) consisting of 20 mM sodium phosphate (pH 7.5) and 150 mM NaCl, and 100 μl was added to a plate surface-treated with streptavidin to fix doxorubicin. . The plate was left at room temperature for 1 hour and then washed six times with PBS buffer (PBST) containing 0.1% Tween-20.

<1-2> biopanning

We used a biopanning method to find proteins that bind doxorubicin. First, 100 μl of a T7 phage library (Novagen) containing human liver cDNA was added to the plate, and the reaction was slowly shaken at room temperature for 40 minutes. The plate was washed 10 times with PBST and then treated with 1% sodium dodecyl sulfate (SDS) for 30 minutes to elute the phages bound to the plate. Eluted phages were amplified by infection with E. coli BLT5615 (Novagen). The amplified phages were used as the next biopanning step. In the second, third, and fourth biopanning steps, the concentrations of NaCl and Tween-20 were increased to 170, 200, 250 mM, 0.3, 0.5, and 0.7%, respectively. 2.5 mM biotin was used. The number of phages in the eluted solution was 10 ⁷ pfu / ml (pfu: plaque forming unit) at the first biopanning, but increased to 10 ⁹ pfu / ml at the fourth biopanning, indicating that phages that specifically bind to doxorubicin are affected by biopanning. Increased by. Changes in the number of phages in the final wash of each stage of biopanning and the number of phages in solution are shown in A of FIG. 1 .

The size of cDNA inserted into the eluted phage was examined by PCR. From the second biopanning, DNAs of 600 bp and 870 bp began to appear, and the other PCR products decreased significantly. Results of the PCR product for each step are shown in B and C of Figure 1; B of FIG. 1 directly used a phage eluate, and C of FIG. 1 randomly took plaques and PCR.

<1-3> DNA sequencing

DNA was isolated from the phages eluted in the fourth biopanning and DNA sequences of PCR products having a size of 870 bp among 20 clones were analyzed. The DNA sequence was compared with other DNA sequences in GeneBank using the BLAST program. As a result, the C-terminal region of the human nucleus phosphate protein 140 (hNopp140) (from 388 to 603 of 699 amino acids) was analyzed. It was confirmed that the DNA sequence of. The DNA nucleotide sequence of the C-terminal part of the human nucleus phosphate protein is described by SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 1 . Other clones had stop codons within 20 amino acids of the N-terminal site and were no longer analyzed.

<1-4> Plaque Hybridization

We performed plaque hybridization to determine whether phage expressing the C-terminal region of hNopp140 specifically increased through biopanning. A plate containing about 1,000 plaques was adsorbed onto the nylon membrane, and then plaques conjugated with search DNA (hNopp140C) were detected by ECL (enhanced chemiluminescence, Amersham Pharmacia Biotech.) Method. It was confirmed that the number of plaques detected was increased. The results of plaque hybridization are shown in FIG. 2 .

Example 2 Expression and Purification of the C-terminal Site Protein of hNopp140

cDNA insertion from T7 phage (T7-hNopp140C) containing the C-terminal part of hNopp140 was amplified by PCR, cleaved with Bam HI and Hind III, and the thioredoxin gene was inserted into pET28a expression vector (Novagen). The pTrx expression vector was cloned. The plasmid was transformed into Escherichia coli BL21 (DE3) (Novagen) and incubated in LB medium containing ampicillin at a concentration of 0.1 mg / ml.

When the culture solution of Example 2 reached an absorbance of 0.7 at 600 nm, E. coli was recovered by adding 1 mM IPTG for 3 hours and then centrifuging. The cells were lysed with a French Presser, and then the Ni-NTA column and the hydroxyapatite column were sequentially performed to purify the protein. The purified protein was dialyzed with 10 mM Mops (3- (N-morpholino) propanesulfonic acid) buffer solution (10 mM Mops, 100 mM NaCl, 1 mM EDTA), concentrated and stored, and named "Trx-hNopp140C". It was. The amino acid sequence and base sequence of Trx-hNopp140C are set forth in SEQ ID NOs: 3 and 4 , respectively. The protein has a thiorredoxin protein linked to the N-terminal portion of the C-terminal region protein of hNopp140. The Trx-hNopp140C has a cleavage site of a protease thrombin between thioredoxin and hNopp140C, but since the protein was continuously cleaved after thrombin treatment, the Trx-hNopp140C showed no Trx without the thrombin cleavage. -hNopp140C was used. The purified protein is shown in lane 1 of Figure 3 times A.

Example 3 Phosphorylation of Proteins

The inventors confirmed whether the hNopp140C of the present invention has the same phosphorylation properties as the original hNopp140. hNopp140 has been reported to be phosphorylated to a very high degree by casein kinase II (Pai et al., J. Cell. Sci ., 1995, 108, 1911-1920; Chen and Yeh, Biochem. Biophys. Res. Commun ., 1997, 230, 370-375). Therefore, it was confirmed whether the purified Trx-hNopp140C also has the same phosphorylation properties as hNopp140. First, 5 μg of protein was mixed with 50 U of casein kinase II and 20 μl of kinase buffer (20 mM Tris-HCl, 50 mM KCl, 10 mM MgCl ₂ , 2.5 mM ATP, pH 7.5). After the reaction, the mixture was phosphorylated by reaction at 30 ° C. for 2 hours. To dephosphorylation, 5 U of calf intestine alkaline phosphatase and 5 μg of protein were mixed with 20 μl of reaction solution (5 mM Tris-HCl, 10 mM MgCl ₂ , 10 mM NaCl, 0.1 mM dithiothreitol, pH 7.9) and then reacted at 37 ° C. for 1 hour. Treated proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Urea-PAGE.

As a result, the original state and dephosphorylated Trx-hNopp140C showed the same mobility in SDS-PAGE and Urea-PAGE, but in the case of phosphorylated protein, the molecular weight increased by phosphate groups than the original protein on SDS-PAGE. Due to the much slower movement speed, ³² P-ATP was also observed at the same position when phosphorylation was used ( A in FIG. 3 ). For a phosphorylated protein, rather it was found that it has moved more quickly because of Urea-PAGE in the negative charge of the phosphate group increases (B in Fig. 3). The results indicate that the protein expressed in Escherichia coli maintains the original form of hNopp140.

Example 4 Enzyme Linked Immunosorbent Assay

We tested the binding specificity of doxorubicin to T7-hNopp140C and Trx-hNopp140C by ELISA. Phage particles adhered to doxorubicin-coated 96-well plates were detected by ELISA using a T7-Tag antibody (Novagen) conjugated with horseradish peroxidase (HRP) diluted in PBS 1/1000. In the case of Trx-hNopp140C, a protein expressed in E. coli, which was mass-expressed and isolated through an expression vector, anti-thioredoxin antibody (Sigma) was diluted by 1/2000 in PBS, and then detected. Phage solution and Trx-hNopp140C solution was added to the plate coated with doxorubicin 0.1 ㎖ reacted at room temperature for 1 hour and washed 6 times with PBST aqueous solution. The anti-thioredoxin antibody was added to the anti-rabbit antibody (Sigma, 1/2000 dilution in PBS), a secondary antibody conjugated with HRP, and reacted at room temperature for 1 hour, followed by washing 6 times. After washing, each plate was reacted for 3-5 minutes by adding 0.1 ml of an aqueous solution of peroxidase substrate containing 1 mg / ml of OPD (o-Phenylenediamine dihydrochloride), and then stopped by adding 0.1 ml of 2.5 M sulfuric acid. The absorbance of was measured at 492 nm.

As a result, T7-liver (T7 phage library containing human liver cDNA) showed a stronger positive signal for T7-hNopp140C since it was selected by biopanning, which resulted in the selected T7-hNopp140C phage surface. hNopp140C is shown. On the other hand, the positive signal of Trx-hNopp140C was much stronger than thioredoxin itself. From this result, it can be seen that the C-terminal protein of hNopp140 has specific binding to doxorubicin ( FIG. 4 ).

In the above results, the positive signal of T7-hNopp140C is lower than that of Trx-hNopp140C. The reason for this is that since the antibody used is different, the magnitude of the signal cannot be compared to the same degree, and a small amount of hNopp140C protein appears on the surface of the T7 phage. This is because the signal will be relatively weaker than the Trx-hNopp140C containing hNopp140C.

Experimental Example 5 Analysis of Adhesion of Doxorubicin to C-terminal Site Protein of hNopp140

We investigated direct interactions between doxorubicin and the C-terminal region protein of hNopp140 using fluorescence spectroscopy and binding affinity was determined using a BIA core biosensor analyzer. Fluorescence emission spectra by the protein of doxorubicin were mixed with 0.7 μM doxorubicin in 10 mM Mops pH 7.5 solution containing 150 mM NaCl and 0.5 mM EDTA, followed by excitation at 490 nm and 530 The emission spectra from nm to 600 nm were obtained and compared.

The result showed the maximum emission spectrum at 555 ㎚ Trx-hNopp140C by confirms the strength increase of the emission spectrum was by interesting phosphorylated Trx-hNopp140C is large change in the fluorescence spectrum was not (Fig. 5 A ) .

In biosensor analysis, doxorubicin was immobilized for 6 minutes at a flow rate of 5 μl / min on a CM5 sensor chip activated with EDC / NHS (N '-(3-dimethylaminopropyl carbodiimidehydrochloride / N-hydroxysuccinimide). The protein samples were inactivated by the addition of M ethanolamine Protein samples were diluted in working buffer (10 mM Mops (pH 7.5), 150 mM NaCl, 1 mM EDTA) and flowed at a flow rate of 30 μl / min. Gram was identified and dissociation sensorgrams were checked by flowing the working buffer solution, using cells completely deactivated with ethanolamine only to calibrate and correcting for nonspecific binding by subtracting the sensorgram of ethanolamine from the sensorgram of doxorubicin. .

As a result, Trx-hNopp140C showed strong binding signal and dissociation constant with doxorubicin was calculated to be 4.52 × 10 ^-6 M. On the other hand, when only Trx was flowed, no binding signal could be confirmed. Therefore, the binding signal between doxorubicin and Trx-hNopp140C was due to hNopp140C. As in fluorescence analysis, phosphorylated Trx-hNopp140C showed no binding to doxorubicin ( B of FIG. 5 ).

As described above, the C-terminal region protein of hNopp140 of the present invention has a strong binding ability with the anticancer drug doxorubicin in the cell, so it is possible to understand the mechanism of anticancer activity of doxorubicin in vivo and to reduce the side effects of doxorubicin and the anticancer effect. It can be useful to improve.

<110> Korea Institute of Science and Technology <120> C-terminus protein of human nucleolar phophoprotein hNopp140 and the gene ana <130> 1p-05-29 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 216 <212> PRT <213> Homo sapiens <400> 1 Ser Asn Lys Pro Ala Val Thr Thr Lys Ser Pro Ala Val Lys Pro Ala 1 5 10 15 Ala Ala Pro Lys Gln Pro Val Gly Gly Gly Lys Leu Leu Thr Arg 20 25 30 Lys Ala Asp Ser Ser Ser Ser Glu Glu Glu Ser Ser Ser Ser Glu Glu 35 40 45 45 Glu Lys Thr Lys Lys Met Val Ala Thr Thr Lys Pro Lys Ala Thr Ala 50 55 60 Lys Ala Ala Leu Ser Leu Pro Ala Lys Gln Ala Pro Gln Gly Ser Arg 65 70 75 80 Asp Ser Ser Ser Asp Ser Asp Ser Ser Ser Ser Glu Glu Glu Glu Glu 85 90 95 Lys Thr Ser Lys Ser Ala Val Lys Lys Lys Pro Gln Lys Val Ala Gly 100 105 110 Gly Ala Ala Pro Ser Lys Pro Ala Ser Ala Lys Lys Gly Lys Ala Glu 115 120 125 Ser Ser Asn Ser Ser Ser Asp Asp Ser Ser Glu Glu Glu Glu Glu 130 135 140 Lys Leu Lys Gly Lys Arg Ser Pro Arg Pro Gln Ala Pro Lys Ala Asn 145 150 155 160 Gly Thr Ser Ala Leu Thr Ala Gln Asn Gly Lys Ala Ala Lys Asn Ser 165 170 175 Glu Glu Glu Glu Glu Glu Glu Lys Lys Lys Ala Ala Val Val Val Ser Lys 180 185 190 Ser Gly Ser Leu Lys Lys Arg Lys Gln Asn Glu Ala Ala Lys Glu Ala 195 200 205 Glu Thr Pro Gln Ala Lys Lys Ile 210 215 <210> 2 < 211> 648 <212> DNA <213> Homo sapiens <400> 2 tcaaataagc cagctgt cac caccaagtca cctgcagtga agccagctgc agcccccaag 60 caacctgtgg gcggtggcca gaagcttctg acgagaaagg ctgacagcag ctccagtgag 120 gaagagagca gctccagtga ggaggagaag acaaagaaga tggtggccac cactaagccc 180 aaggcgactg ccaaagcagc tctatctctg cctgccaagc aggctcctca gggtagtagg 240 gacagcagct ctgattcaga cagctccagc agtgaggagg aggaagagaa gacatctaag 300 tctgcagtta agaagaagcc acagaaggta gcaggaggtg cagccccttc caagccagcc 360 tctgcaaaga aaggaaaggc tgagagcagc aacagttctt cttctgatga ctccagtgag 420 gaagaggaag agaagctcaa gggcaagcgc tctccaagac cacaagcccc caaggccaat 480 ggcacctctg cactgactgc ccagaatgga aaagcagcta agaacagtga ggaggaggaa 540 gaagaaaaga aaaaggcggc agtggtagtt tccaaatcag gttcattaaa gaagcggaag 600 cagaatgagg ctgccaagga ggcagagact cctcaggcca agaagata 648 <210> 3 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> Trx- hNopp140C <400> 3 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Ala 1 5 10 15 Ala Gly Ser His Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser 20 25 30 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe 35 40 45 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp 50 55 60 Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 65 70 75 80 Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 85 90 95 Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 100 105 110 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu 115 120 125 Ala Gly Ser Gly Ser Gly Asp Asp Asp Asp Lys Val Pro Leu Val Pro 130 135 140 Arg Gly Ser Met Leu Gly Asn Pro Asn Ser Ser Asn Lys Pro Ala Val 145 150 155 160 Thr Thr Lys Ser Pro Ala Val Lys Pro Ala Ala Ala Pro Lys Gln Pro 165 170 175 Val Gly Gly Gly Gln Lys Leu Leu Thr Arg Lys Ala Asp Ser Ser Ser 180 185 190 Ser Glu Glu Glu Ser Ser Ser Ser Glu Glu Glu Lys Thr Lys Lys Met 195 200 205 Val Ala Thr Thr Lys Pro Lys Ala Thr Ala Lys Ala Ala Leu Ser Leu 210 215 220 Pro Ala Lys Gln Ala Pro Gln Gly Ser Arg Asp Ser Ser Ser Asp Ser 225 230 235 240 Asp Ser Ser Ser Ser Glu Glu Glu Glu Glu Lys Thr Ser Lys Ser Ala 245 250 255 Val Lys Lys Lys Pro Gln Lys Val Ala Gly Gly Ala Ala Pro Ser Lys 260 265 270 Pro Ala Ser Ala Lys Lys Gly Lys Ala Glu Ser Ser Asn Ser Ser Ser 275 280 285 Ser Asp Asp Ser Ser Glu Glu Glu Glu Glu Lys Leu Lys Gly Lys Arg 290 295 300 Ser Pro Arg Pro Gln Ala Pro Lys Ala Asn Gly Thr Ser Ala Leu Thr 305 310 315 320 Ala Gln Asn Gly Lys Ala Ala Lys Asn Ser Glu Glu Glu Glu Glu Glu 325 330 335 Lys Lys Lys Ala Ala Val Val Val Ser Lys Ser Gly Ser Leu Lys Lys 340 345 350 Arg Lys Gln Asn Glu Ala Ala Lys Glu Ala Glu Thr Pro Gln Ala Lys 355 360 365 Lys Ile Lys Leu Ala Ala 370 <210> 4 <211> 1125 <212> DNA <213> Artificial Sequence <220> <223> Trx-hNopp140C <400> 4 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtggcggc cggcagccat 60 atgagcgata aaattattca cctgactgac gacagttttg acacgg atgt actcaaagcg 120 gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 180 ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 240 atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 300 ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 360 aaagaattcc tcgacgctaa cctggccggt tctggttctg gtgatgacga tgacaaggta 420 cccctggttc cgcgtggatc gatgctcggg aatccgaatt cttcaaataa gccagctgtc 480 accaccaagt cacctgcagt gaagccagct gcagccccca agcaacctgt gggcggtggc 540 cagaagcttc tgacgagaaa ggctgacagc agctccagtg aggaagagag cagctccagt 600 gaggaggaga agacaaagaa gatggtggcc accactaagc ccaaggcgac tgccaaagca 660 gctctatctc tgcctgccaa gcaggctcct cagggtagta gggacagcag ctctgattca 720 gacagctcca gcagtgagga ggaggaagag aagacatcta agtctgcagt taagaagaag 780 ccacagaagg tagcaggagg tgcagcccct tccaagccag cctctgcaaa gaaaggaaag 840 gctgagagca gcaacagttc ttcttctgat gactccagtg aggaa gagga agagaagctc 900 aagggcaagc gctctccaag accacaagcc cccaaggcca atggcacctc tgcactgact 960 gcccagaatg gaaaagcagc taagaacagt gaggaggagg aagaagaaaa gaaaaaggcg 1020 gcagtggtag tttccagatc agattgagga agacgaga

Claims (10)

C-terminal region protein of humane nucleolar phospheprotein (hereinafter referred to as "hNopp140") having the amino acid sequence set forth in SEQ ID NO: 1 and having the property of binding to doxorubicin. delete The gene of the C-terminal region protein of hNopp140, encoding the amino acid sequence set forth in SEQ ID NO: 1 . The gene according to claim 3, wherein the gene has a nucleotide sequence set forth in SEQ ID NO: 2 . A protein having an amino acid sequence set forth in SEQ ID NO: 3 and having a thiorredoxin bound to the N-terminus of the C-terminal region protein of hNopp140. delete A gene of a protein in which thioredoxin is bound to the N-terminus of the C-terminal region protein of hNopp140, which encodes the amino acid sequence of SEQ ID NO: 3 . 8. The gene according to claim 7, wherein the gene has a nucleotide sequence set forth in SEQ ID NO: 4 . Expression vector pTrx-hNopp140C comprising the gene of claim 8. Escherichia coli transformant transformed with the expression vector of claim 9 E. coli BL21 (DE3)