KR100379577B1 - Cell-transducing HIV-1 Tat-glutamate dehydrogenase fusion protein, polynucleotide coding the fusion protein, expression vector of the fusion protein and the transducing method of the fusion protein - Google Patents

Abstract

Genetically unified TAT-GDH fusion by fusing human glutamic acid dehydrogenase (GDH) gene with 9 amino acid human immunodeficiency virus type-1 TAT protein transduction site (RKKRRQRRR) using bacterial vector Obtained protein. The TAT-GDH fusion protein thus prepared was efficiently infiltrated into PC12 cells by Western blot analysis and enzyme activity after incubation. Denatured TAT-GDH fusion proteins penetrated into cells more efficiently than undenatured. The infiltrated TAT-GDH fusion protein showed the complete activity of GDH in the cell, and the nine TAT protein transduction sites attached to the N-terminus of GDH had no effect on GDH activity or thermal stability.

Description

Cell penetrating teeage-human glutamic acid dehydrogenase fusion protein, recombinant polynucleotide encoding the fusion protein, expression vector of this fusion protein, and method for introducing the fusion protein into cells {Cell-transducing HIV-1 Tat-glutamate dehydrogenase fusion protein, polynucleotide coding the fusion protein, expression vector of the fusion protein and the transducing method of the fusion protein}

The present invention relates to a fusion protein of a cell penetrating human glutamate dehydrogenase (hereinafter abbreviated herein as "GDH") with TAT, a recombinant polynucleotide encoding the fusion protein, an expression vector of the fusion protein, and TAT- The present invention relates to a method of introducing a GDH fusion protein into a cell. More specifically, a recombinant protein is used to prepare and purify a fusion protein in which nine HIV-1 Tat proteins are bound to the N-terminal side of human GDH and into the cell with high efficiency. It relates to human GDH that is permeated and has activity in cells.

GDH is an enzyme that promotes the interconversion of L-glutamate and 2-oxoglutarate using NAD ⁺ or NADP ⁺ as coenzymes (Smith, E., Austin, B.). , Blumenthal, K. and Nyc, J. (1975) in: The Enzymes (Boyer, P., ed.) Vol. 11, pp. 293-367, Academic Press, New York.McPherson MJ and Wootton JC (1983) Nucleic Acids Res. 11, 5257-5266.Cho S.-W., Lee J. and Choi SY (1995) Eur.J. Biochem. 233, 340-346.Rice, DW, Hornby, DP and Engel, PC ( 1985) J. Mol. Biol. 181, 147-149. Veronese, FM, Nyc, JF, Degani, Y., Brown, DM and Smith, EL (1974) J. Biol. Chem. 249, 7922-7928). The importance of GDH is well demonstrated in neurodegenerative diseases caused by a lack of GDH activity. GDH is also closely related to hypoglycemia and hyperinsulinism.

At least four GDH isomers are known to exist in the human cerebrum (Duvoisin RC, Chokroverty S, Lepore F and Nicklas WJ (1983) Neurology 33, 1322-1326. Plaitakis, A., Berl, S. and Yahr, MD (1984) Ann. Neurol. 15,144-153. Hussain, MH, Zannis, VI and Plaitakis, A. (1989) J. Biol. Chem. 264, 20730-20735), these GDH isomers are distributed differently (Plaitakis). , A., Flessas, P., Natsiou, AB, and Shashidharan, P. (1993) Can. J. Neurol. Sci. Suppl. 3, S209-S116). On the other hand, there has been an interesting report that GDH isomers isolated from some patients with multisystem atrophy are deficient (Hussain, M. H., Zannis, V. I. and Plaitakis, A. (1989)). The source of this GDH polymorphism (poloymorphysm) has not been identified yet, but four different sizes of mRNAs present in human brain tissue have been reported (Plaitakis, A., Flessas, P., Natsiou, AB, and Shashidharan, P). (1993)). Recently, cDNAs of GDH expressing genes without introns linked to the X chromosome have been identified (Shashidharan P., Michaelidis ™, Robakis NK, Kresovali A., Papamatheakis J., and Pliatakis A. (1994)). J. Biol. Chem. 269, 16971-16976).

Methods for treating neurodegenerative diseases caused by GDH deficiency include transduction of viral vectors capable of expressing GDH by cell manipulation or introduction of proteins into cells. Several methods have been tried to achieve this goal (Renneisen, K., Leserman, L., Mattes, E., Schroder, HC, and Muller, WE (1990) J. Biol. Chem. 265, 16337-16342. Prior, TI, Fitzgerald, DJ, and Pastan, I. (1991) Cell, 64, 1017-1023.Leamon, CP and Low, PS (1992) Proc. Natl. Acad. Sci. USA. 88, 5572-5576) . However, these methods have several problems such as mass production of proteins, low penetration rate into target cells, and limitations on specific genes or cell types (Leamon, CP and Low, PS (1992) Proc. Natl. Acad. Sci. USA. 88, 5572-5576. Matsumoto, K. and Nakamura, T. (1992) Crit. Rev. Oncol. 3, 27-54). Therefore, the development of technology that directly penetrates the complete form of protein into cells can be a good way to solve these problems.

Recently, the human immunodeficiency virus (Hunan Immunodeficiency Virus type-1; hereinafter abbreviated as "HIV") protein is a transactivator of transcription (TAT) protein (hereinafter referred to as "TAT protein", "TAT", " TAT domain "in the same sense) has been found to efficiently move through the cell membrane and into the cytoplasm easily. 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, specific receptors or carriers do not appear to be involved in the transmembrane TAT protein, and it is understood that the protein transduction site of the 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 to 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 carry 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.

Recently, the present inventors have chemically synthesized the GDH gene capable of expressing human GDH and successfully expressed it in E. coli. The recombinant GDH had activity and had the same characteristics as the GDH purified from tissues.

The present invention seeks to solve the problems of the prior art in introducing or expressing GDH into cells and to introduce them into cells, particularly brain cells or neurons, and to have activity in cells with high efficiency.

In order to achieve the above object, the present inventors invented a new method of integrating TAT protein into chemically synthesized human GDH by genetic engineering and then infiltrating into PC12 cells, which are neuronal cells. This technique of infiltrating GDH into cells in the form of protein is a method that has never been used before, and thus the importance of this technology is very important in that it can be one of new treatment methods such as neurodegenerative diseases caused by the lack of GDH activity. It is considered to be high.

1A and B are DNA sequences of the TAT-GDH gene. The amino acid TAT protein of 9 amino acids was attached between the EcoRI / NruI of the human GDH gene prepared by the present inventors to show a modified part. The numbers on the left are the sequences of amino acids (top) and DNA (bottom). The nine amino acids of the TAT transducing protein, shown in italics, are located -1 to -9 on the amino acid sequence. Factor Xa action sites are indicated by arrows. Amino acid sequence 1 is the first amino acid (Ser) that appears in the sequence unique to human GDH.

2 is a cleavage map of the TAT-GDH fusion protein expression vector.

3 shows the results of SDS / PAGE analysis of TAT-GDH purified from E. coli BL21 DE3 cell extract.

Lane 1: purified TAT-GDH

Lane 2: molecular weight marker proteins.

4 shows the results of SDS / PAGE analysis of PC12 cell extracts transduced with TAT-GDH.

Lane 1: Extract of PC12 cells grown in culture medium with only GDH not fused with TAT.

Lane 2: Extract of PC12 cells grown in culture with TAT-GDH fused to TAT.

5 is The transduction of the recombinant TAT-GDH protein into PC12 cells over time was analyzed by Western blot. TAT-GDH was administered to the culture in the presence of 1 mM chloroquine to infiltrate directly into PC12 cells. After 4, 8, 12, and 24 hours of cell collection and destruction, the extracts were analyzed by Western blot. Monoclonal antibodies used in Western blot analysis were produced by the inventors with the cerebellar tissue GDH. At this time, as a control, extracts of PC12 cells grown in TAT-GDH fused to TAT culture were used for Western blot analysis.M, Size indicating proteins; C , Control; S , TAT-GDH.

The present invention relates to a fusion protein TAT-GDH fused with HIV TAT protein to transport into a cell GDH known to play an important role in the nervous system and to have activity in the cell, a vector for preparing the fusion protein TAT-GDH, Provided is a method for introducing a fusion protein prepared using the vector into cells excluding human cells.

In the present invention, as a way to treat neurological diseases caused by GDH deficiency, a method of infiltrating neurons by fusion of TAT protein of HIV, which is known to be easy to invade cells into chemically synthesized human GDH gene Devised.

In order to make such a TAT-GDH fusion protein, the following method was tried. First, nine amino acids (RKKRRQRRR) present in the TAT protein were placed just before amino acid 1 (Ser) of human GDH. Second, by changing the DNA sequence to the preferred form of E. coli, it was possible to prevent the formation of inclusion bodies while increasing the expression rate. Finally, factor Xa recognition site (Ile-Glu-Gly-Arg) (Nagia, K. and Thogersen, HC (1987) Methods Enzymol. 153, 461-481), a proteolytic enzyme with high specificity, was added. By removing unnecessary amino acids that were inevitably attached in the process of genetic manipulation, the protein as close as possible was obtained.

To this end, a TAT-GDH expression vector was developed that can overexpress and easily purify the TAT-GDH fusion protein. This expression vector (pET-Tat-GDH) contains human GDH, 9 amino acids (TAT 49-57) at the TAT transduction site, and cDNA capable of expressing 6 histidine residues at the amino acid terminal. .

Using this expression vector, the TAT-GDH fusion protein was overexpressed in E. coli and purified by metal-chelating affinity chromatography in both denatured and natural conditions.

The denatured TAT-GDH fusion protein was confirmed by Western blot to be delivered to the cells in time and concentration-dependent manner in cultured HeLa cells. On the other hand, TAT-GDH purified in nature and GDH used as a control protein were not transported into cells.

In cells treated with TAT-GDH fusion protein, the activity of GDH enzyme increased in proportion to the amount of protein carried into the cell. These results indicate that TAT can move GDH into cells and artificially increase the activity of GDH in cells.

The present invention relates to a cell penetrating TAT-GDH fusion protein covalently bonded to the HIV Tat transduction site 49-57 residues (Tat 49-57) to the amino terminus of human GDH or a derivative thereof.

In addition, the present invention is a recombinant polynucleotide encoding the TAT-GDH fusion protein is coupled to the HIV Tat transduction region 49-57 residue (TAT 49-57) coding DNA sequence on the 5 'side of human GDH or derivative cDNA thereof It is about.

The present invention also relates to a vector for expressing a cell penetrating TAT-GDH fusion protein constructed as shown in the restriction map of FIG. 5 including the recombinant polynucleotide to express the fusion protein.

In addition, the present invention comprises the steps of expressing the vector in a microorganism;

Purifying the expressed TAT-GDH fusion protein into denatured state using 8M urea or the like;

The present invention relates to a method of introducing a TAT-GDH fusion protein composed of denatured TAT-GDH fusion proteins into cells, except for human cells.

Large amounts of TAT-GDH were obtained by expressing pTAT-GDH in E. coli DE3. After incubating the E. coli DE3 for 3 hours at 37 ° C in the presence of 1 mM IPTG, the specific activity of GDH in the aqueous extract was 1.15 units / mg. In other words, the TAT-GDH protein is similar to the characteristics of the known human GDH, and thus the nine amino acids of the TAT protein attached to the N-terminus have little effect on the GDH activity. .

The TAT-GDH thus expressed was isolated and purified by a purification method established by the present researchers (Cho S.-W., Lee J. and Choi SY (1995) Eur. J. Biochem. 233, 340-346). . Purified TAT-GDH was treated with factor Xa to remove unwanted N-terminal amino acids of purified TAT-GDH, followed by HPLC Protein-Pak 300SW gel filtration column. Purification again using.

SDS / PAGE of the purified TAT-GDH protein showed a purity of 98% or higher (FIG. 2). The protein size on SDS / PAGE also agrees well with the GDH size and the size predicted in the DNA sequence known in the literature (Cho S.-W., Lee J. and Choi SY (1995) Eur. J. Biochem. 233, 340). -346).

The protein thus obtained was analyzed for the amino acid sequence of the N-terminus using Edman grinding. As a result, the amino acid sequence analysis of the first 12 amino acids (RKKRRQRRRSEA) was in good agreement with the amino acid sequence of the originally designed TAT-GDH protein.

The purified TAT-GDH was infiltrated directly into PC12 cells through the denaturation step as described above in the cell culture medium. According to the SDS / PAGE analysis, the expressed TAT-GDH protein corresponds to about 11% of the total protein of the aqueous extract (FIG. 3). The protein corresponding to TAT-GDH was very weak in the extract of PC12 arrested by the administration of pure GDH that was not fused with TAT, which is analyzed as GDH originally present in PC12 cells themselves. Western blot analysis of TAT-GDH infiltrated into PC12 cells by time period showed that the amount of GDH present in the extracts of PC12 cells treated with TAT-GDH was up to 9 times higher than pure GDH without fusion with TAT. It was found that (Fig. 5).

Comparing the specific activity of PC12 cell extracts, TAT-GDH administration (1.19 units / mg) was 9 times higher than GDH-only administration (0.13 units / mg) (Table 1).

sample Specific activity (unit / mg) C4h 0.13 C8h 0.10 C12h 0.13 C24h 0.13 S4h 0.91 S8h 1.25 S12h 1.52 S24h 1.20

These results show that the TAT-GDH protein has successfully penetrated into PC12 cells and that the infiltrating proteins have full activity. The exact reason for this protein folding is unknown, but perhaps chaperones such as HSP90 (Schneider, C., Sepp-Lorenzino, L., Nimmesgern, E., Ouerfelli, O., Danishefsky, S., Rosen, N) , and Hartl, FU (1996) Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp 90. Proc. Natl. Acad. Sci. USA. 93, 14536-14541. Gottesman, S., Wickmer, S., and Maurizi, MR (1997) Protein quality control: Triage by chaperones and proteases.Genes Dev. 11, 815-823).

One peculiar fact is that denatured TAT-GDH has higher penetration efficiency than undenatured TAT-GDH upon protein infiltration. In other words, the administration of unmodified TAT-GDH to PC12 cells showed only a 1.7-fold increase compared to that of GDH alone, which is much less than the 9-fold rate measured for denatured TAT-GDH. . This denatured TAT-GDH has a higher protein penetration efficiency because it is thought energetically that the denatured and unstructured protein forms are easier to pass through the cell membrane than proteins with tertiary or quaternary structures. .

Pharmaceutical compositions containing the TAT-GDH 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, ie, intravenously, subcutaneously, intraperitoneally, or topically. The dosage may be 0.1-100 mg / kg of the daily dose. 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 Preparation of TAT-GDH Fusion Gene.

Oligonucleotides were purchased from the University of California, San Francisco, Biomolecular Resource Center. Oligonucleotides were purified by denatured complex acrylamide electrophoresis (SDS / PAGE) using 8-12% acrylamide / 8M urea gel, using Waters Sep-Pack cartridges or Pharmacia NAP-25 columns. The salts were removed (Sambrook, J., Fritsch, EF and Manuatis, T. (1989) in: Molecular cloning. Cold Spring Harbor Laboratory Press, New York.). Oligonucleotides were phosphorylated with 50 pmol DNA, 50 mM Tris-HCl (pH 8.O), 20 mM MgCl ₂ , 5 mM dithiothreitol, 5 mM ATP, and 10 units of polynucleotide kinase. The compound was reacted at 37 ° C. for 30 minutes and then treated at 65 ° C. for 15 minutes to stop the reaction. Ligated to the phosphorylated nucleotide 2.5pmol each buffer (ligation buffer; 66 mM Tris- HCl, pH 7.6 / 6.6 mM Cl 2/15 mM The ET Otsu ray Toll / 0.44 mM ATP) mix after 5 minutes heating at 95 ℃ in And slowly cooled to room temperature.

The plasmid, which expresses the TAT-GDH gene (pTAT-GDH), was created by modifying the human GDH gene plasmid that our researchers synthesized chemically. First, the GDH gene was cut with Eco RI and Nru I, and vector DNA was isolated using 1% low melting point agarose. The Eco RI / Nru I portion of the plasmid thus obtained was replaced with an oligonucleotide of 69 bp capable of expressing 9 amino acids (RKKRRQRRR) at the TAT translocation site (FIG. 1).

The recombinant oligonucleotide thus prepared was converted to Escherichia coli BL21 (DE3) by boiling, cooling, and ligation. E. coli BL21 DE3 cells were fractionated in LB agar medium containing 100 mg ampicillin / ml and the selected pTAT-GDH was confirmed by DNA sequencing. Other common DNA manipulation techniques were performed according to the methods in the literature (Sambrook, J., Fritsch, E. F. and Manuatis, T. (1989) in: Molecular cloning. Cold Spring Harbor Laboratory Press, New York.).

Example 2 TAT-GDH Purification and Characterization

DE3 / pTAT-GDH was incubated at 37 ° C. until A ₆₀₀ became 0.5, followed by addition of 1 mM IPTG for 3 hours at 37 ° C. The cell precipitate obtained by centrifugation was dissolved in 50 mM Tris / HCl, pH 7.4, 100 mM NaCl, and 2 mM dithiothreitol, and the cells were disrupted using an ultrasonic mill. Cell extracts obtained through centrifugation were separated and purified by GDH (Cho S.-W., Lee J. and Choi SY (1995) Eur. J. Biochem. 233, 340-346). Purification was carried out purely using the same method.

The TAT-GDH thus obtained was confirmed by SDS-PAGE and western blot. At this time, the monoclonal antibody used in the western blot was used in the laboratory of the present inventors using the cerebellum tissue GDH (Choi, SY, Hong, JW, Song, M.-S., Jeon, SG, Bahn, JH, Lee, BY, Ahn, J.-Y. and Cho, S.-W. (1999) J. Neurochem. 72, 2162-2169). Protein concentration measurements were made using the Bradford method (Bradford, M. (1976) Anal. Biochem. 72, 248-254).

The denatured TAT-GDH used for the transduction into PC12 cells was tested at 90 ° C. in the presence of Tris / HCl, pH 6.8, 1% SDS, 5 mM dithiothreitol, 10% glycerol. After heating for a minute and slowly cooled samples were used after removing the SDS using an Amicon concentrator.

<Example 3> PC12 Cell Culture

PC12 cells were cultured in RPMI 1640 solution containing 10% FBS, 5% horse serum, 100 IU / ml penicillin and 100 mg / ml streptomycin. At this time 5% CO ₂ and 37 ℃ was maintained. Once every 4 days it was changed to RPMI 1640 containing 10% horse serum, 100 IU / ml penicillin, and 100 mg / ml streptomycin. TAT-GDH (25 mg / ml) was incubated for a predetermined time (4, 8, 12, and 24 hours) by directly administering PC12 cells with 1 mM of chloroquine concentration. As a control, human GDH that did not fuse with the TAT signal was administered under the same conditions. Each time, TAT-GDH treated cells were collected and washed twice with PBS, followed by RIPA buffer solution (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg). Cells were ground by dissolving in / ml phenylmethanesulfonic acid, 0.3 trypsin inhibitor unit / ml aprotinin, and 1 mM sodium orthovanadate, pH 7.4. GDH activity and total protein amount were measured using the supernatant obtained by centrifugation at 15,000 × g for 30 minutes, and the degree of protein expression was confirmed by Western blot. The base unit of GDH was defined as the amount of protein capable of oxidizing NADH 1mmole per minute at 25 ° C.

The results are shown in FIG.

The present invention is the first experimental report to permeate human glutamic acid dehydrogenase (GDH) directly into cells at the protein level.

In particular, TAT-GDH administered intracellularly restores activity, suggesting that TAT-GDH can be efficiently used for protein treatment.

Based on the present invention, it is possible to deliver GDH directly into cells, suggesting the possibility that it may be usefully used for the treatment of degenerative neurological diseases due to the lack of GDH activity.

Claims (9)

A cell-penetrating TAT-GDH fusion protein in which the HIV-1 TAT transduction site 49-57 residue (TAT 49-57) is covalently bonded to the amino terminus of glutamic acid dehydrogenase or a derivative thereof. The cell penetrating TAT-GDH fusion protein of claim 1. An HIV-1 TAT transduction site 49-57 residue (TAT 49-57) coding DNA sequence is bound to the 5 'side of glutamic acid dehydrogenase or a derivative cDNA thereof, thereby encoding the TAT-GDH fusion protein of claim 1 or 2. SEQ ID NO: 1 or its equivalent recombinant polynucleotide. A vector expressing a cell penetrating TAT-GDH fusion protein comprising the recombinant polynucleotide of claim 3 to express the fusion protein of claim 1. The vector for expressing the cell-penetrating TAT-GDH fusion protein according to claim 4, wherein the expression vector is configured as shown in the restriction map of FIG. Expressing the vector of claim 4 or 5 in a microorganism; Purifying the expressed TAT-GDH fusion protein in a denatured state; Adding a denatured TAT-GDH fusion protein to cells other than human cells; introducing a TAT-GDH fusion protein into cells other than human cells. 7. The method of claim 6, wherein the TAT-GDH fusion protein is denatured using 8M urea. The TAT-GDH fusion protein of claim 1 or 2 as an active ingredient, characterized in that it comprises a pharmaceutically acceptable carrier, pharmaceutical composition used as a therapeutic agent for GDH dependent diseases. According to claim 8, wherein the GDH-dependent disease is characterized in that hypoglycemia, hyperinsulinemia, neurodegenerative diseases or complex atrophy, TAT-GDH fusion protein as an active ingredient, characterized in that it comprises a pharmaceutically acceptable carrier A pharmaceutical composition used as a therapeutic agent for GDH dependent diseases.