KR100379578B1 - Cell-transducing HIV-1 Tat-C3 transferase fusion protein, expression vector of the fusion protein and the analysing method of Rho protein using the fusion protein - Google Patents

Abstract

The present invention is to introduce a cell-permeable C3 transferase fusion protein with Tat, a recombinant polynucleotide encoding the fusion protein, an expression vector of the fusion protein, and a Tat-C3 transferase fusion protein into cells excluding human cells. As a method, more specifically, a recombinant vector is used 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 the C3 transferase. And C3 transferase to function in cells.

Tat-C3 transferase inhibited the macrophages of macrophages, altered RhoA in cells, and even in vitro experiments, Tat-C3 transferase modified RhoA. In conclusion, Tat-C3 transferase effectively migrates into cells, modifies and inhibits RhoA in cells, and inhibits phagocytosis of RhoA-associated macrophages. Using this, Tat-C3 transferase can be effectively introduced into cells to investigate the function of RhoA in particular cells.

Description

Cell-transducing HIV-1 Tat-C3 transferase fusion protein using cell-penetrating thio-C3 transferase fusion protein, expression vector of fusion protein and thio-C3 transferase , expression vector of the fusion protein and the analysing method of Rho protein using the fusion protein}

The present invention is a fusion protein of a cell permeable C3 transferase (hereinafter referred to as "C3 transferase", "C3" is used interchangeably, all have the same meaning), a recombinant polynucleotide encoding the fusion protein, The expression vector of the fusion protein and the Tat-C3 transferase fusion protein are introduced into cells excluding human cells. More specifically, six histidine residues on the N-terminal side of the C3 transferase using recombinant vectors. The present invention relates to a C3 transferase, which penetrates into cells with high efficiency by producing and purifying a fusion protein in which 9 HIV-1 Tat proteins are bound to each other.

The immune response begins with the macrophages' macrophages. Complement receptors and immunoglobulin receptors are major macrophage receptors against pathogen invasion. Rho protein plays a very important regulatory role in two ways of macrophages. Macrophages by immunoglobulin receptors are regulated by Cdc42 and Rac, and macrophages by complement receptors by RhoA. (Caron and Hall, 1998; Massol et al., 1998). RhoA associated with Ras is ADP-ribosylated by recombinant C3 transferase expressed in E. coli and loses its intrinsic activity. Thus, this action may inhibit the macrophages regulated by RhoA in macrophages. However, C3 transferase does not easily move through the cell membrane into the cell.

Cell function is often regulated by RhoA. In this case, RhoA inhibits RhoA in order to examine specific functions of cells. In this case, it is C3-transferase that specifically inhibits RhoA. However, C3 itself does not easily enter the cell, and commercially available C3 has a disadvantage in that it cannot handle a large amount because the value is too expensive. To date, RhoA's functions are known as follows.

RhoA V14, an active mutation of RhoA, is introduced into fibroblast cells to form stress fibers and focal adhesion (Ridley and Hall, 1992). Rac and Rho are also involved in the regulation of endocytosis. The introduction of active mutations of Rac and Rho into cells inhibits receptor-mediated endocytosis of the transferrin receptor (Lamaz et al., 1996). Rho is also involved in the regulation of the cell cycle. When Rho is inhibited, Ras induces the synthesis of a cyclin-dependent-kinase inhibitor (CDKI) p21Waf1 / Cip1 rather than proliferating the cell, preventing it from entering the DNA synthesizer in the cell cycle. On the other hand, when Rho is activated, p21Waf1 / Cip1 is inhibited by Ras and Ras induces DNA synthesis. Cells lacking p21Waf1 / Cip1 do not require Rho activation for DNA synthesis by Ras. This suggests that once Ras is activated, Rho signaling plays a role in inhibiting p21Waf1 / Cip1 (Olson et al., 1998).

The delivery of proteins into tissues and across the blood-brain barrier is severely limited by their biochemical properties and size. However, HIV Tat proteins have been reported to easily cross cell membranes (Green, M., and Loewenstein). Thus, attempts have been made to combine Tat and proteins into cells. It has been reported that the administration of 120 kDa β-galactosidase protein in combination with HIV's Tat protein transfer protein intraperitoneally delivers β-galactosidase to all tissues in mice. These results open up new possibilities for the direct administration of proteins to patients in protein therapy as well as genetic testing of laboratory animals. Heterologous proteins chemically crosslinked with Tat's 36-amino acid domain can migrate into cells (Frankel, A. D., and Pabo).

Other transduction domains include Drosophila's Antennapedia (ANTp) protein (Derossi et al., 1996) and HSV's HSV VP22 protein. Although the exact transport mechanism across the cell membrane is unclear, the fact that small Antp peptides can enter cells at 4 ° C and 37 ° C without receptors suggests that they can move cell membranes without receptors. We do not yet know how Tat protein enters the cell.

Recently, Tat-RhoA14V and -RhoA19N have been used to analyze the effects of RhoA on the regulation of the osteoskeleton of osteoclasts. Also, streptolysin O was treated to inhibit RhoA. It has been reported that C3 transferase was introduced into cells (Chellaiah et al., 2000). However, streptolysine O can partially degrade a cell and cause the protein in the cell to leak out. Therefore, Tat-C3 transferase is believed to be a safer way to transfer C3 transferase into cells.

The present invention seeks to solve the problems of the prior art in introducing or expressing C3 transferase into cells and to introduce them into cells with high efficiency and to have activity in cells.

In addition, the present invention is to investigate the intracellular function of RhoA by inhibiting RhoA by treating a cell with a small amount of Tat-C3 transferase.

In order to achieve the above object, the present inventors fused the C3 transferase and the protein transduction site of the Tat protein to confirm that the fusion protein was effectively penetrated into cells. In addition, the physiological effects of Tat-C3 transferase in macrophages were investigated.

1 is a cleavage map showing insertion of C3 and Tat-C3 into a pET15b vector.

Plasmid map of pETC3 or pET-Tat C3 (A). pET-C3 and Tat-C3 were identified by agarose gel electrophoresis. Lanes 1, 3: intact plasmids, lanes 2, 4: pET, C3 and Tat-C3 fragments (B). C3 or Tat-C3 was purified by His-bound resin column. And it was confirmed by Western blot (E) using SDS-PAGE (C, D) and anti-C3 antibody.

2 is a Western blot showing the migration of Tat-C3 into J774A.1 macrophages.

20-60 ug / ml Tat-C3 or C3 protein was incubated at 37 ° C for 1 hour in J774A.1 cells (5x10 ⁵ ) in the medium. Proteins on the surface of the cells were removed with trypsin, and only proteins transferred into the cells were identified with SDS-PAGE and anti-His antibodies.

3 is a graph showing the effect of C3 or Tat-C3 on the macrophages of macrophages.

The degree of association of macrophages was determined using a fluorescence spectrophotometer at various times after treatment with C3 (□) or Tat-C3 (■) at 50 ug / ml at 37 ° C. It was confirmed by adding 3mM crystal violet.

4 is an electrophoretic and western blot photograph showing ADP-ribosylation of RhoA in J774A.1 cells.

Macrophages in medium were treated with C3 (50 ug / ml) or tat-C3 (50 ug / ml) for 24 hours at 37 ° C. The suspension of cell lysate was electrophoresed in SDS-PAGE (14% gel) (A) and undenatured PAGE (14% gel) (B). Rho protein was identified by Western blot using anti-RhoA antibody.

FIG. 5 is an electrophoresis and western blot photograph showing ADP-ribosylation of RhoA by Tat-C3 transferase in vitro.

For in vitro assays, 30 ug / ml C3 or Tat-C3, 3 uM NAD +, 6 uM in a total of 50 ul buffer (50 mM triethanolamine HCl, pH 7.5, 2 mM MgCl2, 1 mM DTT and 0.2 mM PMSF) GDP, 60 ng / ml GST-RhoA was mixed on ice. The ADP-ribosylation reaction was started by adding the GST-RhoA protein and incubated at 37 ° C for 1 hour. Rho was analyzed by undenatured PAGE (A) and SDS-PAGE (B) and confirmed by Western blot using anti-RhoA antibody.

6 is a polynucleotide sequence encoding a Tat-C3 fusion protein.

The present invention provides a fusion protein Tat-C3, a fusion protein Tat-C3 fused with HIV-1 Tat protein to transport and function within the cell C3 transferase involved in the macrophages of macrophages A vector, and a method for introducing the fusion protein into cells other than human cells.

To this end, a Tat-C3 expression vector was developed that can overexpress and easily purify the Tat-C3 fusion protein. This expression vector (pET-Tat-C3) contains a C3 transferase, 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-C3 fusion protein was overexpressed in E. coli and purified easily and conveniently by metal-chelating affinity chromatography in denaturing and natural state.

The denatured Tat-C3 fusion protein was confirmed by Western blot to be delivered time- and concentration-dependently to cultured macrophages. On the other hand, Tat-C3 purified in nature and C3 used as a control protein were not transported into cells.

Tat-C3 transferase effectively migrates into cells, modifies and inhibits RhoA in cells and inhibits phagocytosis of RhoA-associated macrophages.

The present invention relates to a cell penetrating Tat-C3 transferase fusion protein covalently bonded to the HIV-1 Tat transduction site 49-57 residue (Tat 49-57) at the amino terminus of C3 transferase or a derivative thereof.

In addition, the present invention is the Tat-C3 transferase fusion protein is coupled to the HIV-1 Tat transduction site 49-57 residue (Tat 49-57) coding DNA sequence is coupled to the 5 'side of the C3 transferase or its derivative cDNA To a recombinant polynucleotide encoding a.

The present invention also relates to a vector expressing a cell penetrating Tat-C3 transferase fusion protein constructed as shown in the restriction map of FIG. 1A 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-C3 transferase fusion protein by denatured using 6M urea;

A method of introducing a Tat-C3 transferase fusion protein comprising a denatured Tat-C3 transferase fusion protein into a cell, but not into human cells.

Pharmaceutical compositions containing the TAT-C3 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, that is, 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.

material

Zymosan A particles and fluorescein isothiosyanate (FITC) were purchased from Sigma chemicals, Anti-RhoA, anti-His and anti-C3 transferases from Santa Cruz, Ni2 + -gels and columns from Novagen. To overexpress GST-RhoA protein, 0.1 mM isopropylthio-β-D-galactoside was added to Escherichia coli (DH5α) transformed with a pGEX2T-RhoA plasmid. GST-RhoA protein expression was induced, then disrupted by sonication, and purified by glutathione-sepharose 4B beads (Pharmacia).

Example 1 Preparation of Expression Vector

PTat-C3 was prepared as follows to express a protein in which the C3 transferase was bound to a major part of HIV1-Tat (amino acids 49-57). First, two oligonucleotides were synthesized to generate two strands of oligonucleotides encoding nine amino acids, the major part of HIV-1 Tat.

Top strand: 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3 ',

Bottom strand: 5'-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3 '.

NisI-XhoI was used to cut the 6His open reading frame region of pET15b (Invitrogen, Carlsbad, Calif.) To connect two oligonucleotides synthesized to prepare a pHisTat expressing HisTat.

The nucleotide sequence of pHisTat was analyzed using a fluorescence-based automated sequencer model 373A (Applied Biosystems, Inc.). Next, a polymerase chain reaction (PCR) was performed using Pfu DNA polymerase (Clontech) on a plasmid having a complete C3 transferase as a nucleotide sequence.

Sense primer: 5'-CTCGAGTATTCAAATACTTACCAGGAG-3 ',

Antisense primer: 5'-GGATCCTTATTTAGGATTGATAGCTGTGCC-3 '.

PCR products were treated with XhoI-BamHI and inserted at the XhoI-BamHI position of the pHisTat vector. The plasmid prepared as above was named pTat-C3. In a similar manner, pC3 was prepared by inserting the XhoI-BamHI treated PCR product into the region of XhoI-BamHI of pET15b.

Clones with the expected inserts were screened by XhoI-BamHI to perform postsequence analysis (Sambrook et al., 1989).

Example 2: Expression and Purification of Tat-C3 Fusion Proteins

To obtain pure C3 and Tat-C3 transferase, C3 and Tat-C3 were cloned into pET-15b plasmid vector and expressed in bacterial cells. Proteins with these His-tags were purified using Ni2 + -columns.

BL21 Escherichia coli (Pharmacia) transformed with pC3 or pTat-C3 were incubated at 37 ° C. in LB containing 100 ug / ml of ampicillin for one day. This was further diluted 10 times with fresh LB and further incubated for 4 hours at 37 ° C. at 260 rpm until OD ₆₀₀ = 1.0. Protein expression was induced by adding IPTG at a concentration of 0.1 mM for 5 hours. 6M urea and protease inhibitors (20 mg / ml soybean trypsin inhibitor, 2 mg / ml aprotinin) It was disrupted by sonication in binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) containing 5 mg / ml leupeptin, 100 mg / ml phenyl methyl sulfonyl fluoride (PMSF). After centrifugation to remove cell debris, the cleared cell extracts were loaded onto a Ni 2+ -IDA column. The column was first washed with binding buffer free of 6M urea and then with wash buffer (80 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl, pH 7.9). Proteins were purified by eluting buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl pH 7.9) and desalted on PD10 column (Amersham). A typical Tat-C3 fusion protein was obtained by adding denaturing agent in the same manner as above.

The protein concentration of each fraction was separated by SDS-PAGE based on bovine serum albumin (BSA) and quantified by densitometric analysis. Protein quantification was quantified with the Bradford Protein Assay Kit (Biorad). Standard used BSA. The purified C3 fusion protein was dissolved in PBS containing 20% glycerol and dispensed in an amount sufficient for use, and then stored at -80 ° C.

Purified C3 and tat-C3 each represent a single band of 35 kDa. Tat-C3 transferase appears on SDS-PAGE as it is slightly larger than the size of C3 transferase with Tat peptide attached (FIG. 1)

Example 3: Cell Culture and Introduction of Tat-C3 Fusion Proteins

To confirm the transfer of Tat-C3 transferase into macrophages, the transferred Tat-C3 was identified by Western blot using an anti-His antibody that recognizes the His-tag portion of the protein. The protein was treated and trypsin was treated to remove the remaining protein without moving into the cell.

Macrophage J774A.1 cell line contains 20 mM HEPES / NaOH (pH 7.4) containing 5 mM NaHCO ₃ , 10% fetal calf serum (FBS) and antibiotics (100 U / ml streptomycin, 100 U / ml penicillin). ) Was incubated in DMEM (Dulbecco's Modified Eagle's Medium) with 5% CO ₂ at 37 ℃. J774A.1 cells were treated with various concentrations of Tat-C3. By time after treatment, cells were detached with trypsin-EDTA (Gibco BRL) and washed three times with PBS (phosphate buffered saline). Cells were harvested to obtain cell extracts for performing Western blot analysis.

Tat-C3 transferase was seen in cells while C3 was not identified in the cells (FIG. 2), indicating that Tat-C3 transferase was easily migrated into cells.

Example 4 Preparation of FITC-zymosan

Zymosan A granules are labeled with FITC, centrifuged, the precipitate is collected, resuspended in FBS (controlled to 5 x 10 ⁸ zymoic acid granules / ml), and then dispensed and stored at 70 ° C. It was.

Example 5 Investigation of Macrophages

Phagocytosis of macrophages was measured to investigate whether the transferred Tat-C3 transferase had normal physiological function. Already C3 transferase inhibits RhoA activity by ADP-ribosylation of RhoA, and macrophages are known to involve RhoA. Phagocytosis measured the intensity of fluorescence of FITC-Zymoic acid incorporated into cells. In addition, the fluorescence of FITC-Zymoic acid adhering to the cell surface without being introduced into the cell was removed by the addition of crystal violet.

Cells were incubated in a 35 mm dish at a density of 2 x 10 ⁵ cells, 5 x 10 ⁵ FITC-conjugated zymosan granules were incubated at 37 ° C. for 30 min, washed three times with PBS and then washed with PBS. It was suspended in 1 ml. FITC was excited at 490 nm and FITC fluorescence was measured immediately using a fluorophotometer at 520 nm. FITC granules adhered to the cells without being introduced into the cells were removed by addition of 3 mM crystal violet.

C3 did not inhibit the phagocytosis of macrophages, whereas Tat-C3 transferase decreased in both zymotic binding and macrophages with macrophages (FIG. 3). Cat transferase bound to Tat inhibited RhoA activity by ADP-ribosylation of RhoA to normal. However, Tat-C3 did not completely inhibit phagocytosis. It is believed that Tat-C3 transferase did not completely inhibit RhoA in cells or involved phagocytosis of other pathways independent of Rho.

Example 6: ADP-ribosylation of RhoA protein by Tat-C3 transferase

To investigate whether Tat-C3 transferase inhibits RhoA's activity by ADP-ribosylation of RhoA, RhoA was analyzed by PAGE or SDS-PAGE from Tat-C3 transferase treated macrophages.

To determine if Tat-C3 transferase directly induces ADP-ribosylation of RhoA, purified GST-RhoA was stored with NAD + and C3-transferase, and the modified RhoA was analyzed by electrophoresis.

Macrophage cells were treated with 10-50 ug / ml C3 or tat-C3 for 24 hours at 37 ° C. Supernatants of cell lysates were analyzed by SDS-PAGE and nondenaturing PAGE (14% gel). Rho protein was confirmed by Western blot using anti-RhoA antibody. For in vitro experiments, 30 ug / ml C3 (or Tat-C3), 3 uM NAD, 6 uM GDP, 60 ng / ml GST-RhoA was buffered (50 mM triethanolamine HCl, pH 7.5, 2 mM MgCl). ₂ , 1 mM DTT, 0.2 mM PMSF) in a total volume of 50ul and stored on ice. The ADP-ribosylation reaction was started by adding the GST-RhoA protein and stored at 37 ° C. for 1 hour. Modified RhoA was identified by Western blot using PAGE and SDS-PAGE not denatured using anti-RhoA antibodies.

RhoA modified with ADP-ribosylation shows fast migration in undenatured PAGE (Med. Enzymol; Sehr et al., 1998). RhoA of Tat-C3 treated cells showed slow migration on PAGE or rapid migration on SDS-PAGE. This means that Tat-C3, like C3, can cause modification of RhoA (FIG. 4).

C3 transferase modified RhoA in the presence of NAD + and altered Rho migration in electrophoresis (FIG. 5). Like C3 transferase, Tat-C3 delayed the migration of SDS-PAGE RhoA and accelerated the movement of RhoA in PAGE. This indicates that arginine residues modified with ADP-ribosylation changed the charge of RhoA (Selkine, et al., 1989).

According to the present invention, C3 transferase at the same concentration as Tat-C3 did not migrate into the cell (FIG. 2), and also did not inhibit the effects of macrophages (FIG. 3), and at the same concentration as Tat-C3 transferase. Did not alter RhoA in cells (Fig. 4).

Tat-C3 transferases, on the other hand, are much more effective than C3 transferases to inhibit RhoA in cells. Tat-C3 transferase inhibited the macrophages of macrophages, altered RhoA in cells, and even in vitro experiments, Tat-C3 transferase modified RhoA. In conclusion, Tat-C3 transferase effectively migrates into cells, modifies and inhibits RhoA in cells, and inhibits phagocytosis of RhoA-associated macrophages.

C3 transferase protein was not easy to move into the cell had a limit to use, but the present invention solved this problem by binding Tat to C3 transferase.

Using this, Tat-C3 transferase can be effectively introduced into cells to investigate the function of RhoA in particular cells.

In addition, the present invention will be useful for the prevention and treatment of diseases involving C3 transferase and RhoA protein.

Claims (7)

A cell-penetrating Tat-C3 transferase fusion protein having covalently linked to HIV-1 Tat transduction site 49-57 residue (Tat 49-57) at the amino terminus of C3 transferase or a derivative thereof. An HIV-1 Tat transduction site 49-57 residue (Tat 49-57) coding DNA sequence is coupled to the 5 'side of a C3 transferase or derivative thereof cDNA to encode the Tat-C3 transferase fusion protein of claim 1 SEQ ID NO: 5 or an equivalent recombinant polynucleotide thereof. A vector expressing a cell penetrating Tat-C3 transferase fusion protein comprising the recombinant polynucleotide of claim 2 to express the fusion protein of claim 1. The vector expressing the cell penetrating Tat-C3 transferase fusion protein according to claim 3, wherein the expression vector is configured as shown in the restriction map of FIG. 1A. Expressing the vector of claim 3 or 4 in a microorganism; Purifying the expressed Tat-C3 transferase fusion protein in a denatured state; Adding a denatured Tat-C3 transferase fusion protein to cells other than human cells; introducing a Tat-C3 transferase fusion protein into cells other than human cells. 6. The method of claim 5, wherein the Tat-C3 transferase fusion protein is denatured using 6M urea. A pharmaceutical composition useful for C3 transferase and RhoA protein-related diseases, comprising the TAT-C3 transferase fusion protein of claim 1 as an active ingredient and a pharmaceutically acceptable carrier.