KR100472937B1 - Cell-transducing HIV-1 Tat-cytosine deaminase fusion protein and use of the fusion protein - Google Patents

Abstract

The present invention is intended to transport cytoplasmic diamines (CDs) known to play an important role in gene directed enzyme / prodrug therapy (GDEPT) into cells and to be active in cells. A fusion protein Tat-CD fused with HIV-1 Tat protein, a polynucleotide encoding the fusion protein Tat-CD, a vector for producing the fusion protein, and a use thereof, wherein the Tat-CD fusion protein is used in pharmaceutical compositions and the like. Useful for use.

Description

Cell-transducing HIV-1 Tat-cytosine deaminase fusion protein and use of the fusion protein

The present invention relates to a fusion protein of Cytosine deaminase (hereinafter referred to herein as "CD" or "yCD", meaning the same), a recombinant polynucleotide encoding the fusion protein, the fusion protein As related to the expression vector and its use, more specifically, by using a recombinant vector to prepare and purify a fusion protein in which six histidine residues and nine HIV-1 Tat proteins are bound to the N-terminal side of cytosine dianaminase The present invention relates to cytosine dianases having the activity of permeating into cells with high efficiency and specifically destroying only cancer cells in cells.

Many chemotherapy that are currently used for cancer therapy do not allow selective treatment of cancer cells. Therefore, recent cancer treatments have been progressing toward inducing death of only cancer cells by selectively delivering a compound (compound) only to cancer cells. Enzyme / prodrug systems using nontoxic prodrugs with the possibility of selectively treating only cancer cells have been proposed, and this method uses specific enzymes of cancer cells to produce prodrugs. By turning to a drug, the cancer cells are killed selectively.

Enzyme / prodrug therapy refers to a method of treatment that induces death of cancer cells or cells in abnormal conditions. The principle is that the enzyme or prodrug itself has no effect on the target cell, but their combination causes the prodrug to turn into a new form of harmful drug, eventually leading to cell death. Enzymes / prodrug systems in recent years have used specific enzymes that cancer cells do not have, and cloned in front of the enzyme genes that want to express promoters that respond to transcription factors present only in cancer cells. As a result, it has been developed to insert cells into target cells and induce cell death. This method is called gene directed enzyme / prodrug therapy ("GDEPT") and many forms of GDEPT have been reported (TA Connors 1995). Enzymes used in GDEPT can only be used if the following criteria are met. First, the enzymes used in GDEPT should be those that the target cell to which the gene is to be inserted does not have. Generally, the enzymes that the cancer cells do not have are used. Second, the enzyme must have a property known as the bystander effect, which refers to the ability to react with other substrates other than the original substrate to produce new harmful products (Ion Niculescu-Duvaz et. al 1998 ).

Cytosine deaminase (hereinafter referred to as "CD") is not a gene of cancer cells and is used in many GDEPTs as an enzyme having properties of foreign effect. CD is an enzyme involved in the deamination of cytosine. CD, an enzyme found in many microorganisms, is involved in the metabolism of cytosine and is known to convert harmless prodrugs, 5-FC (fluorocytosine) into very harmful drug 5-FU (fluorouracil) by bystander effects (FIG. 8). , Richard Jund et al 1970 , P. Erbs et al 2000 ). Cytosine, unlike other nucleobases, is not only directly degraded to ammonia by cellular enzymes but also used in salvage synthesis, where cytosine is hydrolyzed by CD to uracil. (Kim, Tae Hyun et al 1998 ).

The killing of 5-FU is currently found to be due to several types of mechanisms (Philippe Erbs et al 2000 ). 5-FU is known to inhibit protein synthesis and DNA synthesis by changing to various derivatives. As a representative mechanism, the transformed 5-FU is transformed into 5-FUTP and 5-fluoro-dUTP (5-FdUTP) forms by enzymes in the cell and is involved in cell death. 5-FdUTP inhibits DNA replication by inhibiting an enzyme called thymidylate synthase, inducing a deficiency of dTTP used for DNA replication. On the other hand, another derivative, 5-FUTP, is known to directly participate in messenger RNA synthesis and inhibit the synthesis of normal proteins (Jacob Kream et al 1952 , Mark S. Khil et al 1996 , Mullen CA et al 1992 ).

CD is present in many microorganisms, especially E. coli and yeast CD has been studied a lot. Yeast CD enzymes are more capable of converting 5-FCs to 5-FUs than bacterial CD enzymes, but stability at high temperatures (37 ° C.) is known to be very low compared to bacterial CDs (Katauragi et al 1989 ).

Recently, gene-derived enzyme / prodrug therapy (GDEPT) using CD genes has been studied (Els Kievit et al 1999 ). In gene induced enzyme / prodrug therapy using CD, when the CD gene is transfected into target cells, the CD protein is expressed to convert 5-FC into 5-FU. 5-FU is transformed into 5-FUTP or 5FdUTP by the enzyme of the cell, which inhibits the expression of protein and the replication of DNA, leading to cell death (Denis Evoy et al 1991 ). Nevertheless, gene-derived enzyme / prodrug therapies have some problems. The insertion efficiency of genes is very low and leads to the expression of persistent and permanent proteins. In addition, there is a disadvantage that can not control the expression level of the protein. This study was conducted to develop a new type of therapeutic method that overcomes these problems and to directly infiltrate purified enzymes into cells rather than transfection with DNA. However, normal proteins cannot cross hydrophobic cell membranes.

Recently, the Tat (transactivator of transcription) protein, a type of Human Immunodeficiency Virus type-1 protein, has been found to efficiently move through the cell membrane and into the cytoplasm. This function appears due to the nature of the protein transduction domain, which is the intermediate region of the Tat protein, and its exact mechanism is still unknown [Frankel, A.D. and Pabo, C. O. (1988) Cell 55, 1189-1193. Green, M. and Loewenstein, P. M. (1988) Cell 55, 1179-1188. Ma, M. and Nath, A. (1997) J. Virol. 71, 2495-2499. Vives, E., Brodin, P. and Lebleu, B. (1997) J. Biol. Chem. 272, 16010-16017.]. However, it is understood that no specific receptors or carriers are involved in the transmembrane of Tat protein and that the protein transduction site of Tat protein occurs by directly interacting with the lipid bilayer of the membrane [Vives, E., Brodin, P. and Lebleu , B. (1997) J. Biol. Chem. 272, 16010-16017. Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. and Prochiantz, A. (1996) J. Biol. Chem. 271, 18188-18193.].

In recent studies, heterologous proteins such as ovalbumin, β-galactosidase, and horseradish peroxidase were fused with HIV-1 Tat protein in vivo tissue and culture. It is shown to be directly transported into the cells of the cells. Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, LL, Pepinsky, B. and Barsoum, J. (1994) Proc. Natl. Acad. Sci. USA 91, 664-668. Schwartze, S.R., Ho, A., Vocero-Akbani, A. and Dowdy, S. F. (1999) Science. 285, 1569-1572. Watson, K. and Edward, R.J. (1999) Biochem. Pharmacol. 58, 1521-1528. These results indicate that the Tat protein has the ability to transport not only itself but also other giant proteins of its kind into the cell.

However, not all proteins are carried by the Tat protein. In addition, it is not yet clear whether all proteins carried into cells by Tat show biological activity.

In the present invention, in order to overcome the problems of gene-derived enzyme / prodrug therapy (GDEPT) by transfection (mixed with "transfection" herein), it is mixed with a protein transduction site ("PTD"). (PTD-DEPT) has been invented by direct transduction of CD enzymes into cells (PTD-DEPT), and the purpose of the present invention is to provide a protein or therapy that exhibits anti-cancer effects without side effects by introducing Tat-CD fusion enzymes. do.

CD genes were fused with 9 amino acids of Tat (RKKRQRRR), cloned into an E. coli expression vector, and the fusion protein was purified purely to introduce the fusion protein into HeLa cells. Anticancer effects caused by the introduced CD fusion protein were observed using enzyme and MTT assays.

The present invention is directed to HIV-induced cytosine deaminase (CD), which is known to play an important role in gene directed enzyme / prodrug therapy, to be intracellular and active in the cell. The present invention provides a fusion protein Tat-CD fused with a 1 Tat protein, a vector for producing the fusion protein Tat-CD, a method for producing and purifying a fusion protein using the vector. The present invention also provides a pharmaceutical composition using the fusion protein Tat-CD.

To this end, a Tat-CD expression vector was developed that can overexpress and easily purify the Tat-CD fusion protein. This expression vector (pTat-CD) contains a human cytosine diaminase, nine amino acids (Tat 49-57) at the Tat transduction site, and a cDNA capable of expressing six histidine residues at the amino acid terminus. Doing.

Using this expression vector, the Tat-CD fusion protein was overexpressed in E. coli and purified easily and conveniently by metal-chelating affinity chromatography.

Tat-CD fusion protein of the present invention can be prepared by a general chemical binding method in addition to gene recombination method.

Tat-CD fusion protein was confirmed by Western blot to be delivered to cells in time and concentration-dependent manner in cultured HeLa cells.

The activity of PK enzyme in cells treated with Tat-CD fusion protein increased in proportion to the amount of protein carried into the cells. These results indicate that Tat can move PK into cells and artificially increase the activity of intracellular CD.

The present invention is a cell-penetrating Tat-cytosine deminase of SEQ ID NO: 7 or a functional equivalent thereof having an HIV-1 Tat transduction site 49-57 residue (Tat 49-57) covalently attached to the amino terminus of cytosine deaminase. It relates to a fusion protein.

In addition, the present invention is bound to the 5 'side of the cytosine diaminase cDNA HIV-1 Tat transduction site 49-57 residues (Tat 49-57) coding DNA sequence is coupled to the Tat-cytosine diamine fusion protein It relates to a recombinant polynucleotide which is SEQ ID NO: 6 or a functional equivalent thereof.

In addition, the present invention relates to a vector expressing a cell penetrating Tat-cytosine deminase fusion protein comprising the recombinant polynucleotide to express the fusion protein, for example, as shown in the restriction map of FIG.

Furthermore, the present invention relates to a pharmaceutical composition comprising the Tat-cytosine deminase fusion protein as an active ingredient and a pharmaceutically acceptable carrier, characterized in that it is used as a therapeutic agent for cancer. The present invention relates to a pharmaceutical composition comprising a Tat-cytosine deminase fusion protein as an active ingredient and a pharmaceutically acceptable carrier. The present invention relates to a Tat cytosine deminase fusion protein amino acid sequence of SEQ ID NO. And the polynucleotide sequence encoding the Tat cytosine deminase fusion protein of SEQ ID NO: 6. For example, as defined by Sambrook et al. (Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2ed.Vol. 1. pp. 101-104, Cold Spring Harbor Laboratory Press (1989)), the present invention relates to Tat cytosine dia. It will be clearly understood that such DNA or RNA sequences are also included that can hybridize to minase fusion protein coding polynucleotide sequences. Thus, the polynucleotide molecules interpreted by the present invention are hybridized to the polynucleotide sequences of the present invention. Inductively from Tat cytosine deminase fusion protein coding polynucleotide sequences or amino acid sequences prepared by the above-described method, as well as nucleic acid molecules having sequences by the degeneracy of the coding sequence and the genetic code. Molecules with inferable sequences are also included. The present invention relates to Tat cytosine diami Izu fusion protein amino acid sequence (SEQ ID NO: 7), as well as, also includes the same sequence as a functional These substituted with other amino acid residues in the sequence in accordance with the silent change. Silent change means that one or more amino acids in the sequence may be substituted with other amino acid (s) of similar polarity that function functionally equivalently. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar, hydrophobic amino acid classes include alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline, and methionine. Polar, neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged, basic amino acids include arginine, lysine and histidine. Negatively charged, acidic amino acids include aspartic acid and glutamic acid. That is, "functional equivalent" of a gene sequence means gene sequences that are translated into the same amino acid sequence, and also refers to the "functional equivalent" of an amino acid sequence. Equivalent "means, for example, amino acid sequences in which the amino acids in the sequence can be substituted with other amino acids of similar polarity that function functionally equivalent to perform the equivalent function.

As used herein, the term "transport", "penetration", "transport", "delivery", "transmission", "pass", "transduction", "cell introduction" for "introduction" of cytosine demininase protein into cells. It is mixed with the expressions.

Pharmaceutical compositions containing a Tat-CD fusion protein as an active ingredient can be formulated orally or by injection by conventional methods in combination with a carrier that is conventionally acceptable in the pharmaceutical art. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g. lactose, textose, sucrose, mannitol, sorbitol, cellulose and / or glycine), suspending agents (e.g. silica, Talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols, and the tablets also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpi It is preferred to contain a disintegrant (eg starch, agar, alginic acid or its sodium salt) or a boiling mixture and / or absorbents, colorants, antiseptics and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries (eg, preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure). In addition, they may contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally as desired, or parenterally, i.e., intravenously, subcutaneously, intraperitoneally, or topically, and the dosage may be from 0.001 to 100 mg / kg. It can be administered in 1 to several times. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Hereinafter, the configuration of the present invention will be described in detail by examples. However, the scope of the present invention is not limited to the following examples.

Example 1.pTat-CD Vector Preparation

pET-yCD, pTat-yCD expression vectors were made using the pET15-b vector (Invitrogen, Carlsbad, U.S.A). As used herein, yCD refers to yeast-derived cytosine diamines. In the examples, experiments were carried out using yeast-derived cytosine deminase enzymes, but it is clear that the scope of the present invention is not specifically limited to yeast-derived enzymes.

The pET15-b vector has a T7 promoter, a Lac operator, and a T7 polyadenylation signal. The pTat expression vector was made as follows. Two DNA oligomers encoding the HIV-1 Tat basic domain to construct a pTat vector (upper chain, 5'-TAG GAA GAA GCG GAG ACA GCG ACG AAG AC-3 '; lower chain, 5'-TCG AGT TCC CGT CGC TGT CTC CGC TTC TTC C-3 '), and the DNA sequence has restriction enzyme cleavage sites of NdeI and XhoI at both ends, respectively. Both DNA oligomers were annealed in double-strand through incubation at 70 ° C. for 30 minutes. After pET15-b was cut with restriction enzymes of NdeI and XhoI , a pTat vector was prepared by inserting a Tat base domain into an open reading frame. Insertion of the Tat gene was confirmed using a fluorescence-based automated sequencer (model 373A; Applied Biosystems, Inc.). And yCD ( FCY1 ) gene was inserted after Tat base domain.

The yCD gene was amplified using PCR to insert the yCD gene. Genomic DNA of Saccharomyce cerevisiae was used as a template for amplification of yCD. dog body for the amplification of yCD gene (primer) 5 having the XhoI cleavage site followed by the 5'-CTC GAG GTG ACA GGG GGA ATG GCA AGC-3 upper strand and the termination codon (stop codon) of "having a XhoI cleavage site Sub-chains of '-CTC GAG CTA CTC ACC AAT ATC TTC-3' were used.

Pfu DNA polymerase was used for PCR, and the template was denatured at 95 ° C for 5 minutes, followed by 30 cycles of 94 ° C 40 seconds, 60 ° C 2 minutes, 50 ° C 1 minute, and amplification further at 60 ° C for 10 minutes. The amplified DNA was cut into XhoI and inserted into the pTat vector. In the same way pET-yCD expression vector was made using pET15-b without the HIV-1 Tat base domain.

Example 2. Expression and Purification of Fusion Proteins

Each expression vector was transformed into BL21 E. coli (pharmacia). E. coli expressing each gene was incubated for 3-4 hours at 37 ℃ until the value of OD (Optical Density) ₆₀₀ is 0.6 ~ 0.7 in 50ml of LB. When the value of OD ₆₀₀ was 0.6-0.7, 1 mM IPTG was added and protein expression was induced for 3-4 hours (Park. Jinseu et al 2000 ). Escherichia coli were separated by centrifugation, denatured on ice for 1 hour in 6M urea buffer, and supernatants centrifuged at 14000 rpm were loaded on a Ni ²⁺ -NTA column (Novagen Ins). . The column was washed with 25 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH7.9) and 25 ml of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). After washing with 4 ml of Isolation Buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH7.9). And the fusion protein purified to remove imidazole was loaded onto PD-10 column using PBS (phosphate saline buffer) (Hikaru Nagahara et al 1998 ). The expressed fusion protein was quantified with a Biorad assay kit.

Expression vectors were made using the pET15-b vector and expressed in E. coli (BL21) (FIG. 1). And purified by denaturation in 6M urea (FIG. 2A). As shown in FIG. 2, the purified protein was separated by 12% SDS-PAGE and stained with coomassie blue. The purified protein was analyzed by immunoblot to confirm that the desired fusion protein was correct (FIG. 2B). Proteins isolated in the same manner as in FIG. 2A were transferred to nitrocellulose membranes, which were measured using an ECL assay kit (FIG. 2B). All fusion proteins were found to be purely purified. Purified protein was imidazole removed through PD-10 desalting column (Amersham) and the concentration of protein was measured.

Example 3. Cell Culture and Introduction of Fusion Proteins

All HeLa cells used in this experiment were incubated in DMEM medium (Dulbecco's modified eagle's medium) containing 10% fetal bovine serum (FBS), 100 μg / ml streptomycin and 100 U / ml penicillin at 37 ° C., 7% CO ₂ . .

To confirm the transduction ability of Tat-yCD, HeLa cells were plated in ⁶ × 10 ⁵ cells / 35mm plates. When about 70% confluent, cells were washed twice with 2 ml of serum-free DMEM and replaced with fresh 1 ml of DMEM. To confirm concentration-induced cell introduction, various concentrations of purified CD or Tat-CD fusion proteins were treated in the medium at 37 ° C. for 2 hours. 1 μM of the fusion protein was treated at 37 ° C. for various periods of time to confirm the cell introduction over time.

In order to confirm the stability in cells, 1 μM fusion protein was treated for 2 hours, washed twice with serum-free media, replaced with fresh medium, and harvested in each cell for each hour. The stability of the was measured.

Cells for measuring all transduction were treated with 500 μl of 0.05% trypsin / EDTA (GIBCOBRL) for 10 minutes at 37 ° C. to remove any remaining fusion proteins outside of the cells after the protein treatment and before harvesting. The cells were then harvested after washing with cold PBS. For Immunoblot, cells were dissolved in lysis buffer (150 nM NaCl, 0.02% sodium azide, 100 μg / ml PMSF, 1% Triton X-100, 50 nM Tris-HCl, pH 8.0.). Incubate at 4 ° C. for 30 minutes.

Example 4 Immunoblot Analysis

Tat-yCD was separated by 15% SDS-PAGE and transferred to nitrocellulose membranes (S and S. Ins). The membrane was blocked for 1 hour with skim milk powder. The membrane was washed twice with 10 ml of TBST (20 mM Tris, 500 mM NaCl, 0.05% Tween-20) for 10 minutes and then incubated with rabbit antibody directed against the tagged histidine (Santa Cruz) for 2 hours. After washing twice with 10 ml of TBST for 10 minutes, it was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Sigma chemical) for 2 hours. Membranes washed twice more for 10 min with 10 ml TBST were detected by manufacturer's instructions (ECL, Amersham).

The ability of Tat-PTD was measured because the purified fusion protein had to migrate into cells to be applied to PTD-DEPT. Enzymes introduced into cells were identified by immunoblotting using six histidines tagged with pET15-b (FIG. 4). First, the efficiency of transduction over time was measured. Tat-yCD was treated with cells at a concentration of 1 μM to measure transduction efficiency over each time. As shown in FIG. 4A, Tat-yCD was measured from 15 minutes after the fusion protein was treated in the culture medium, and almost 90 minutes of all proteins moved into the cell after 90 minutes (FIG. 4A).

In the experiment for measuring the transduction efficiency according to the concentration, the cells were measured by treating various concentrations of fusion proteins for 2 hours. Referring to B of FIG. 4, as the amount of the treated fusion protein increases, the amount of the protein moving into the cell also increases. And yCD fusion proteins without Tat were treated under the same conditions but could not migrate into cells and no bands could be identified. As a result, it was concluded that Tat-PTD effectively introduced fusion protein into cells.

For effective protein cancer therapy using PTD-DEPT, the fusion protein that enters the cell must remain stable for a long time in the cell, and is precisely refolded by chaperone in the cell. Enzyme activity should be obtained. First, it was measured how stable the introduced fusion protein was maintained in the cell (Fig. 4C). Tat-yCD was stable for up to 24 hours in the fusion protein introduced for 2 hours.

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Example 5 Measurement of CD Enzyme Activity For protein therapy with Tat-PTD, transduced Tat-CD fusion proteins should have CD activity. In order to confirm that the fusion protein migrated into the cell has CD enzyme activity, the enzyme activity was measured after transducing the fusion protein into the cell. Fusion proteins were treated in the same way to measure cell transduction efficiency. If the fusion protein moves into the cell, the inactivated fusion proteins will have the correct conformation by the chaperones in the cell and will have enzymatic activity. As shown in FIG. 7A, the cells not treated with the fusion protein could not convert 5-FC into 5-FU, and no changes were found in the cells treated with the fusion protein without Tat. Nevertheless, it can be seen that cells into which the Tat-yCD fusion protein is introduced convert 5-FC into 5-FU. In addition, CD activity is increased in proportion to the concentration of the protein treated in the cell. In the study of the time when the fusion protein moved into the cell had enzymatic activity, the Tat-yCD was treated 180 minutes after the protein was treated. Has maximum activity (FIG. 5B). Comparing this result with the result of introduction (maximum at 90 minutes), it can be seen that a slight error occurs in time. In the case of TaT-yCD, as shown in FIG. Lasts up to time. Taken together, the results show that PTD-DEPT using Tat-CD fusion protein is possible. The enzymatic activity of Tat-yCD in transduction (combined with "transduction") has been improved. The method presented in the paper was measured with a slight modification (Shigeki Kuriyma et al 1999). In order to measure the activity of the introduced enzyme, the cells were processed and harvested according to concentration and time in the same manner as the transduction for identifying immunoblots. Cells were dissolved in 100 μl elution buffer, and then 170 μl PBS (pH 7.4) and 30 μl 5-FC (30 mM, Sigma chemical.) Solution were incubated at 37 ° C. for 24 hours. After 24 hours, 1 ml of 0.1N HCl was added to 50 µl of reaction solution. The CD enzyme activity was measured at 290 nm and 255 nm using a UV absorber (Uvikon 930, Kontron, Ins.), Respectively.

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Purified in the absence of urea to determine the activity of the purified enzyme. 279 μl PBS (pH 7.4) and 30 μl 5-FC (30 mM) solution were added to 1 μg of purified protein, followed by incubation at 37 ° C. for 24 hours. After 24 hours, 1 ml of 0.1N HCl was added to 50 µl of reaction solution. CD enzyme activity was measured at 290 nm and 255 nm using a UV absorber (Uvikon 930, Kontron, Ins.). Activity was obtained by calculating the values measured at 290nm and 255nm with the equation

5-FC [mmol / l] = (0.119 XA ₂₉₀ -0.025 XA ₂₅₅ )

5-FU [mmol / l] = (0.185 XA 255 - 0.049 XA 290)

The activity of the fusion protein was measured using a spectrophotometer for the loss of 5-FC and the resulting 5-FU by deamination of 5-FC. The loss of 5-FC was measured at 290 nm and the production of 5-FU was measured at 255 nm, respectively. As a result of the enzymatic activity, it was found that the fusion protein converts almost all 5-FCs into 5-FU (FIG. 3). yCD and Tat-yCD showed almost the same 90% enzymatic activity as the intact enzyme. However, it can be seen that the fusion protein purified by denaturation with 6M urea has no activity under the same conditions. As a result of the CD enzyme activity, the expressed and purified fusion protein can function intact even when Tat is fused, and when denatured, all of its activity is lost.

Example 6 Cytotoxic Assay

MTT (3- [4,5-dimethlthiazol-2-yl] -2,5-diphenol- to measure the cytotoxic effects of prodrug-to-drug changes by transduced Tat-CD protein tetrazoline bromide, Sigma chemical.) assay was performed. To measure cytotoxicity, cells were plated in 24 well plates with 1 × 10 ⁴ cells each. The next day, washed with serum-free medium and 0.5ml of complete medium was added. Purified CD enzyme and Tat-CD enzyme or 5-FC were treated in 0.5 ml medium for each condition. Medium was removed every 24 hours and fresh medium was treated with protein and 5-FC. The viability of the cells was 96 hours after the addition of 1ml of DMEM containing MTT and incubated for 3 hours at 37 ℃, MTT removal and 1ml isopropanol (isopropanol) was added gently shake for 1 minute and then ELISA OD was measured at 570 nm using a reader.

First, cells were treated with various concentrations of protein in the presence of 100 μM 5-FC. As shown in FIG. 6A, response curves for 5-FC of HeLa cells were confirmed. The cells responded for the first time when treated with 0.5 μg / ml Tat-yCD, and showed viability inversely proportional to the amount of protein treated until 2 μg / ml. And at 2 µg / ml or more, the same reaction was observed.

We also observed how sensitive the transduced cells are to 5-FC. Cells were treated with 5 μg / ml fusion protein and 5-FC at different concentrations. Referring to B of FIG. 6, Tat-yCD reacts from a 5-FC concentration of about 0.5 mM or more. And when the concentration of 5-FC exceeds 2mM showed no effect. In all experiments, the CD fusion protein without the Tat domain was treated together as a control, and only 5-FC was treated. They had no effect on cell growth or death.

Example 7 Bystander cytotoxic effect assay

A study was conducted on the mechanism by which cells are killed by activated drug (5-FU). To measure the biochemical function of 5-FU, cells were plated in two 24-well plates under the same conditions (1 × 10 ⁴ cells / plate). One plate was treated with protein and 5-FC in the same way as it was treated to determine the cytotoxic effect. The other plate was not treated with any protein or chemicals. The rest of the plate used the following method to measure the bystander cytotoxic effects. In order to measure the effect of cell destruction, the culture solution should be changed every 24 hours. The resulting solution was centrifuged and placed in a new plate. This procedure was repeated every 24 hours and after 96 hours the viability of the cells was measured at 570 nm using an ELISA reader via an MTT assay.

The killing of normal cells by the drug activated by the transduced fusion protein was observed as an MTT assay. In Figure 7, it can be seen that once activated drug has a similar effect on the non-transduced cells. However, cell death by activated drugs did not look as strong as the cytotoxic effects of direct transduced cells. As a result of the Bystander cytotoxic effects, the concentration of the cell death mechanism is similar to the apoptosis effect, but does not seem to have the maximum effect.

The present invention is the first experimental report to permeate cytosine deaminase (CD) directly into cells at the protein level.

In particular, Tat-CD administered intracellularly showed activity in cells in a concentration and time dependent manner.

In addition, the present invention suggests that the fusion protein Tat-CD is involved in cell destruction in mammalian cancer cells, and that Tat-CD can be effectively used for protein treatment.

According to the present invention, Tat-CD can be effectively used for protein treatment of diseases by administering CD, which plays an important role in cancer treatment, into cells.

1 shows a schematic structure of plasmid vectors in the present invention.

The vectors are based on pET15-b and have six histidine residues at the N-terminus. Tat-CD fusion proteins are covalently linked to the protein transduction site of Tat and the protein. pTat-CD and pyCD expression vectors were generated by fusion in the yCD coding sequence structure.

Figure 2 shows the expression and purification of yCD, Tat-yCD, bCD and Tat-bCD fusion protein in E. coli. The fusion proteins expressed in E. coli were purified in the form of denaturation with Ni-NTA column, respectively.

(A) Coomassie staining gel of purified yCD and Tat-yCD.

(B) Results of western blot of the gel shown in (A).

Figure 3 is a graph measuring the activity of purified yCD, Tat-yCD, bCD and Tat-bCD in the non-denatured native form.

100 ml of enzyme (1 mg) was added to 170 ml PBS and 30 ml of 30 mM 5-FC solution and incubated at 37 ° C. for 24 hours. Then 50 ml of this solution was stopped with 1 ml of 0.1N HCl. Activity was measured by UV absorbers at 290 nm and 255 nm. de is a protein denatured with 6M urea; control was a mixture of PBS, 5-FC and 0.1N HCl; The other is a mixture of PBS, 5-FC, fusion protein and 0.1N HCl, respectively.

4 is a PAGE result showing the transduction rate of the denatured Tat fusion protein.

Fusion protein was added to the culture medium of HeLa cells under various conditions. The cells lysates were immunoblotted with Levit anti-histidine antibodies after washing the cells twice with serum-free medium before obtaining the cells under various conditions.

C (control) is a non-transducted cell lysate.

(A) shows the time course of transduction. Cells were transduced with 1 mM Tat-CD fusion protein for the indicated time.

(B) shows the degree of concentration dependent transduction. Cells were treated with various concentrations of Tat-CD fusion protein and CD protein for 2 hours under standard conditions.

(C) shows the stability of the transduced Tat-CD fusion protein. Cells were incubated with 1 mM Tat-CD fusion protein for 2 hours, then cells were washed with serum free medium and fresh medium added. The treated cells were then obtained at the indicated time.

5 is a graph measuring the activity of the Tat fusion protein transduced into HeLa cells.

Cells were transduced with fusion proteins and obtained under the conditions identified in FIG. 3. 100 ml of cell lysates transduced with Tat fusion protein were added to the enzyme activity section described, and activity was measured by UV absorbers at 290 nm and 255 nm.

C; A mixture of HeLa cell lysate, PBS, 5-FC and 0.1N HCl; Others are a mixture of PBS, 5-FC, transduced HeLa cell lysate and 0.1N HCl.

(A) Activity measurement result of the transduced enzyme by concentration. Cells were transfected with the fusion protein for 2 hours at standard conditions.

(B) graph of results of activity measurement of transduced enzymes over time. Cells were treated with 80 mg or 50 mg of Tat fusion protein for various times.

(C) Graph of stability measurement results of transduced enzyme activity.

Figure 6 is a graph showing the results of the MTT assay for confirming the biological activity of the transfected enzyme in the presence of 5-FC.

Sensitivity of cells to various types of drugs was determined using an MTT dye reduction assay. Cells were plated at 1 × 10 ⁴ cells / well in 24-hole plates. The next day the medium was removed and cells were treated daily with either fusion protein or 5-FC in 0.5 ml medium. Living cells were measured 96 hours later using an ELISA reader at 570 nm and 550 nm.

(A) HeLa cells were transfected with the indicated fusion proteins and cultured in the presence of 100 mM 5-FC.

(B) HeLa cells were transfected with 5 mg / ml fusion protein and cultured in the presence of various concentrations of 5-FC.

FIG. 7 is a graph showing the results of an MTT assay for proving the exogenous cytotoxic effect of activated drug 5-FU by transduced Tat-CD enzyme.

To confirm biological activity, cells were treated with various fusion proteins, prodrugs, and 5-FC for 24 hours. Other cells replicated according to plating rules. The cell was not treated with any protein or chemical. The medium of untreated cells was exchanged with medium incubated with fusion protein and 5-FC for 24 hours. After 72 hours, the viability of the cells was measured using an ELISA reader at 570 nm and 550 nm.

(A), (B) HeLa cells were transfected with the indicated fusion proteins and cultured in the presence of 100 mM 5-FC.

8 shows a biochemical pathway in which cytosine dianaminase exhibits a cytotoxic effect.

<110> CHOI, SOO YOUNG PARK, JINSEU LEE, hak-joo RYU, ji-yoon <120> Cell-transducing HIV-1 Tat-cytosine deaminase fusion protein and use of the fusion protein <130> WJTJ-TATcd <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Human immunodeficiency virus type 1 <400> 1 taggaagaag cggagacagc gacgaagac 29 <210> 2 <211> 31 <212> DNA <213> Human immunodeficiency virus type 1 <400> 2 tcgagttccc gtcgctgtct ccgcttcttc c 31 <210> 3 <211> 27 <212> DNA <213> Saccharomyces cerevisiae <400> 3 ctcgaggtga cagggggaat ggcaagc 27 <210> 4 <211> 24 <212> DNA <213> Saccharomyces cerevisiae <400> 4 ctcgagctac tcaccaatat cttc 24 <210> 5 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 5 Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 <210> 6 <211> 511 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence which consists of HIV Tat coding sequence and yeast cytosine cDNA sequence <220> <221> CDS (222) (2) .. (508) <400> 6 t agg aag aag cgg aga cag cga cga aga ctc gag atg gtg aca 43 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Val Thr 1 5 10 ggg gga atg gca agc aag tgg gat cag aag ggt atg gac att gcc tat 91 Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp Ile Ala Tyr 15 20 25 30 gag gag gcg gcc tta ggt tac aaa gag ggt ggt gtt cct att ggc gga 139 Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro Ile Gly Gly 35 40 45 tgt ctt atc aat aac aaa gac gga agt gtt ctc ggt cgt ggt cac aac 187 Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg Gly His Asn 50 55 60 atg aga ttt caa aag gga tcc gcc aca cta cat ggt gag atc tcc act 235 Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu Ile Ser Thr 65 70 75 ttg gaa aac tgt ggg aga tta gag ggc aaa gtg tac aaa gat acc act 283 Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys Asp Thr Thr 80 85 90 ttg tat acg acg ctg tct cca tgc gac atg tgt aca ggt gcc atc atc 331 Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly Ala Ile Ile 95 100 105 110 atg tat ggt att cca cgc tgt gtt gtc ggt gag aac gtt aat ttc aaa 379 Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val Asn Phe Lys 115 120 125 agt aag ggc gag aaa tat tta caa act aga ggt cac gag gtt gtt gtt 427 Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu Val Val Val 130 135 140 gtt gac gat gag agg tgt aaa aag atc atg aaa caa ttt atc gat gaa 475 Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe Ile Asp Glu 145 150 155 aga cct cag gat tgg ttt gaa gat att ggt gag ta g 511 Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu 160 165 <210> 7 <211> 169 <212> PRT <213> Artificial Sequence <400> 7 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Val Thr Gly Gly 1 5 10 15 Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp Ile Ala Tyr Glu Glu 20 25 30 Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro Ile Gly Gly Cys Leu 35 40 45 Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg Gly His Asn Met Arg 50 55 60 Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu Ile Ser Thr Leu Glu 65 70 75 80 Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys Asp Thr Thr Leu Tyr 85 90 95 Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly Ala Ile Ile Met Tyr 100 105 110 Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val Asn Phe Lys Ser Lys 115 120 125 Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu Val Val Val Val Asp 130 135 140 Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe Ile Asp Glu Arg Pro 145 150 155 160 Gln Asp Trp Phe Glu Asp Ile Gly Glu 165

Claims (7)

A cell penetrating Tat-cytosine deminase fusion protein of SEQ ID NO: 7 having an HIV-1 Tat transduction site 49-57 residue (Tat 49-57) covalently attached to an amino terminus of cytosine deaminase. The cell penetrating Tat-cytosine diminase fusion protein according to claim 1, wherein several histidine residues are bound upstream of the 49-57 residues (Tat 49-57) of the HIV-1 Tat transduction site. HIV-1 Tat transduction site 49-57 residue (Tat 49-57) coding DNA sequence is coupled to the 5 'side of the cytosine diaminase cDNA fusion of the Tat-cytosine deminase of claim 1 or 2 SEQ ID NO: 6, or a functional equivalent thereof, encoding a protein. A vector for expressing a cell penetrating Tat-cytosine deminase fusion protein comprising the recombinant polynucleotide of claim 3 to express the fusion protein of claim 1. The vector expressing the cell penetrating Tat-cytosine deminase fusion protein according to claim 4, wherein the expression vector is configured as shown in the restriction map of FIG. delete A pharmaceutical composition comprising the Tat-cytosine diaminase fusion protein of claim 1 or 2 as an active ingredient and a pharmaceutically acceptable carrier and used as an anticancer agent.