KR101106262B1 - Transmembrane Delivery Peptide Originated from Human Homeodomain Gene and Protein Transduction Vector - Google Patents

Abstract

The present invention relates to a protein transduction domain and a protein transduction vector using the same, and more particularly, to a protein transduction domain having an amino acid sequence represented by SEQ ID NO: 1 derived from human homeo protein HOXA5 and a protein transduction vector using the same.

According to the present invention, the protein of interest can be efficiently introduced into the cell, which is not only useful for the delivery of drugs consisting of proteins, nucleic acids or macromolecules, but also because it is a protein-derived domain derived from humans, it can be safely used without causing immunity problems.

Cell membrane, peptide, fusion protein, vector, protein transduction domain

Description

Transmembrane Delivery Peptide Originated from Human Homeodomain Gene and Protein Transduction Vector}

The present invention relates to a protein transduction domain and a protein transduction vector using the same, and more particularly, to a protein transduction domain derived from humans and a protein transduction vector using the same, which can effectively introduce proteins, DNA, peptides or macrochemicals into cells. It is about.

Human genes consist of DNA and usually contain information that makes specific proteins. Every cell in the body has the same gene in the chromosome of the cell nucleus, and each cell copies only the selected gene into the mRNA, which serves as a model for making proteins. If a particular gene is mutated, it can't produce a protein or its function decreases. These defects can disrupt the function of cells and tissues and cause disease.

In the past, only drug treatments have been attempted for diseases caused by these inherited genetic mutations. However, in the early 1970s, when they learned about the function of genes and discovered genes related to many genetic diseases, they learned that gene therapy was possible for congenital and acquired diseases. In addition, attempts have been actively made to apply the deficient protein itself as a protein medicine for therapeutic use. The protein has a three-dimensional structure, so the specificity is very high, it is possible to develop drugs with high efficacy and very low side effects by the protein itself. In particular, protein new drugs using intracellular transcriptional factors and signal mediators are expected to be able to develop new drugs with higher efficacy and lower side effects by controlling intracellular signal pathways. However, even if gene therapy or antibiotic replacement protein drug product development is successful, the effectiveness of these macromolecules will not be effective unless they are inhaled into cells.

In gene therapy, in order to transport normal genes to defective cells, viruses that have been removed from pathogenic paths are usually used as a carrier medium. Since viruses have evolved over hundreds of millions of years to transfer their genetic material into host cells, the use of these viruses as gene delivery vehicles must be a fairly powerful technique. However, since humans also have a defense mechanism to remove foreign material, efforts to deliver genes in viral vectors have been challenged by the immune response of these host cells.

The protein transduction domain called PTD (protein transduction domain) is a small peptide consisting of 10-16 basic amino acids, also called cell permeable peptide (CPP) and membrane translocating sequences (MTS). PTD, through the plasma membrane, together with the substances that bind to itself or themselves together with the help of special receptors, accumulates in the cells, effectively transferring proteins, DNA, peptides, or macrochemicals, which were difficult to transfer into cells, into cells. It can be applied. In the case of using PTD, it is possible to transfer a substance in a signal pathway directly into a cell or a nucleus, thereby enabling more specific signal transmission.

PTDs known to date include HIV-1-derived tat proteins, Drosophila-derived Antennapedia homeo domains, HSV-derived transcription factors (VP22) and synthetic lysine / algidine peptides. However, all existing PTDs are peptides derived from or synthesized from external proteins such as viruses, and may cause immune responses when applied to humans. Therefore, it is required to develop a new human-derived PTD that can replace the PTD known to date.

Accordingly, the present inventors found that some amino acid sequences of the human homeo protein HOXA5 may play a role of PTD, and produced a recombinant vector encoding the same, while expressing a fusion protein of the target protein and HOXA5-PTD using the vector. The present invention was completed by confirming the function of PTD.

The present invention provides a novel human-derived protein introduction domain, a gene encoding the domain protein, a gene encoding the gene and the target protein, which can deliver a target protein into cells without fear of inducing an immune response when applied to an actual human body. It is an object to provide a fused recombinant vector.

The present invention for achieving the above object relates to a protein transduction domain (PTD) HOXA5-PTD having an amino acid sequence (RQIKIWFQNRRMKWK) as set forth in SEQ ID NO: 1 derived from human homeo protein HOXA5 and a gene encoding the same. In particular, the gene preferably has a nucleotide sequence of SEQ ID NO.

The present invention also relates to a recombinant vector comprising the gene. The gene encoding the EGFP protein was inserted into the vector to prepare a HOXA5-PTD-EGFP fusion protein. As a result, the EGFP protein itself was not introduced into the cell, whereas the fusion protein was introduced into the cell depending on the concentration and processing time. It was confirmed that the domain protein of the present invention acts as PTD in the fusion protein. In the case of PTD derived from virus, there is a possibility of inducing an immune response in vivo, but the vector of the present invention is expected to be able to rule out this possibility with a PTD derived from humans.

A fusion protein of the target protein and PTD according to the present invention by inserting a gene encoding a protein of interest into the multi-cloning site (MCS) into the recombinant vector and then preparing and expressing a transformant therefrom. Can be expressed. In this case, the target protein can be delivered directly into the cell or the nucleus, thereby enabling more specific signal transmission.

As described above, according to the protein introduction domain of the present invention, the protein of interest can be efficiently introduced into cells, which is useful for the delivery of drugs consisting of proteins, nucleic acids, or macromolecules, and is a protein-derived domain derived from humans. It can be used safely.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

Example

Example 1 Human Genetic Factor Search

In order to search for PTD having a human genetic factor sequence, the amino acid sequence RQIKIWFQNRRMKSK (SEQ ID NO: 3) of PTD of Fushi tarazu (yellow fruit fly) was searched by the Basic Local Aligment Search Tool (NCBI Blast) of Pubmed. As a result, it was determined that the RQIKIWFQNRRMKWK (SEQ ID NO: 1) region of the human homeo protein HOXA5 having 94% homology with the PTD is likely to function as a PTD. The gene encoding the amino acid sequence is represented by SEQ ID NO: 2 in the 770-817 base sequence of HOXA5.

Example 2 Preparation of HOXA5-PTD Expression Vector

To prepare a HOXA5-PTD expression vector, a HOXA5-PTD gene factor (AGACAAATTAAAATCTGGTTCCAAAACCGGAGAATGAAGTGGAAAAAA) of SEQ ID NO: 2 was inserted into an existing E. coli protein vector, pET28b (Novagen) or pET21b (Novagen).

First, oligomers of SEQ ID NOs: 4 and 5, wherein restriction enzyme cleavage sites of NdeI and BamHI were introduced at the N-terminus and C-terminus of the 48-nucleotide HOXA5-PTD gene, respectively. The synthesized two oligomers were denatured at 95 ° C. for 1 minute in a reaction solution containing 50 mM NaCl, and the temperature was slowly lowered to room temperature and regenerated as a double helix sequence. Thereafter, the pET-28b vector or pET-21b vector was digested with restriction enzymes NdeI and BamHI, and then the oligomers regenerated by the double helix sequence were inserted. Expression vectors constructed using the pET28b vector and the pET21b vector by the above method were named pHOXJOIN-N1 and pHOXJOIN-C1 vectors, respectively.

SEQ ID NO: 4'-ata tga gac aaa tta aaa tct ggt tccaaa acc gga gaa tga agt gga aaa aac gg-3 '

SEQ ID NO: 5'-gat ccc gtt ttt tcc act tca ttc tcc ggt ttt gga acc aga ttt taa ttt gtc tc-3 '

The vector prepared by the above method was subjected to electrophoresis on 1% polyacrylamide gel to separate cDNA, confirm DNA sequence, and analyze using GENETYX program. Figure 1 shows the results of the electrophoresis Figure 2 and Figure 3 shows the cleavage map of the pHOXJOIN-N1 vector and pHOXJOIN-C1 vector, respectively. Since the expression vector can be easily produced by the above process using a known gene and a known vector, a separate deposit is omitted.

Example 3 Confirmation of Cell Permeability of HOXA5-PTD

1) Preparation of HOXA5-PTD-EGFP Expression Vector

To determine whether the HOXA5-PTD vector is introduced into the cell, a HOXA5-PTD-EGFP expression vector containing six histidines and a HOXA5 transduction site (RQIKIWFQNRRMKWKK) and fluorescent EGFP was prepared using a pHOXA5-PTD expression vector. It was.

First, the EGFP gene was amplified from the pEGFPN1 vector (Clontech, USA) by PCR using primers of EGFP specific SEQ ID NOs: 6 and 7 containing EcoRI / Xhol restriction enzyme sequences (polymerase chain reaction conditions; 95 ° C, 30 ° C). Sec, 62 ° C. 1 minute, 72 ° C. 2 minutes, 30 cycles).

SEQ ID NO: 5'-gga att cga tgg tga gca agg gcg ag

SEQ ID NO: 7'-ccg ctc gag ctt gta cag ctc gtc

Using the EcoRI, XhoI restriction enzyme (TaKaRa), the pHOXJOIN-N1 vector or pHOXJOIN-C1 vector prepared in Example 2 was cut, and then the pET28b / HOXA5-PTD-EGFP vector and pET21b / HOXA5 were inserted by inserting the amplified EGFP sequence. -PTD-EGFP vectors were completed respectively.

The vectors were transformed into E. coli BL21 (DE3), respectively, to prepare E. coli transformants BL21 (DE3) / pET28b / HOXA5-PTD-EGFP and BL21 (DE3) / pET21b / HOXA5-PTD-EGFP. BL21 (DE3) / HOXA5-PTD-EGFP is characterized by containing six histidine genes for protein purification, HOXA5-PTD genes and EGFP genes.

2) Isolation and Purification of Recombinant HOXA5-PTD-EGFP Fusion Protein

Each of the E. coli transformants prepared in 1) was plated in LB medium containing Ampicillin (50 μg / ml) and incubated at 37 ° C. for 14-16 hours. The following process is a process of separating and purifying the fusion protein from each transformant. Transformed colonies were selected and added to 10 ml of LB medium containing Ampicillin and seed-cultured at 37 ° C., followed by A600 value in 1 L of LB medium containing Ampicillin (50 μg / ml). It was incubated at 37 ℃ until 0.6 ~ 0.8. When the OD value was 0.6-0.8, 1 mM IPTG (isopropylthio-β-D-galactoside) (USB corporation) was added, and further cultured at 25 ° C. for 16 hours to induce expression.

The cultured cells were collected by centrifugation, and then 100 ml of buffer solution (500 mM NaCl, 50 mM Tris pH8.0) was added, and then disrupted by ultrasound (SONICS, VCX-500). The mixture was centrifuged again and the supernatant was taken and loaded onto a column (Bio-rad, Cat No: 732-1010) filled with 5 ml of Ni-NTA agarose (Qiagen, cat No: 30210). After washing with a buffer containing 50 mM imidazole, the fusion protein was eluted with an elution buffer containing 500 mM imidazole.

4A is Coomassie blue staining photograph showing the result of purifying HOXA5-PTD-EGFP fusion protein expressed using each transformant (lane 1: pET28b / HOXA5-PTD-EGFP, lane 2: pET21b /). It can be seen that the molecular weight of HOXA5-PTD-EGFP) and HOXA5-PTD-EGFP fusion protein is about 32 kDa. The purified fusion protein was confirmed to be HOXA5-PTD-EGFP fusion protein once again by Western blot analysis (B of FIG. 4), and was consistent with the Coomassie blue staining result.

The eluted fusion protein was desalted on PD-10 columns (GEHealthcare cat No: 17-0851-01) to remove salts. The salt-free purified HOXA5-PTD-EGFP fusion protein was used while stored at -70 ° C. Coomassie blue staining was performed using SDS-PAGE and compared with bovine serum albumin to calculate the protein concentration, and then diluted according to the concentration used in the examples. In the following examples, only the results of experiments using HOXA5-PTD-EGFP expressed from the E. coli transformants of pET21b / HOXA5-PTD-EGFP were described, but HOXA5- expressed from the transformants of pET28b / HOXA5-PTD-EGFP. PTD-EGFP also showed the same result.

3) Confirmation of introduction of HOXA5-PTD-EGFP fusion protein into cells

(1) Confirmation of introduction into cells

EGFP to confirm the introduction of HOXA5-PTD-EGFP fusion protein into cells And the intracellular influx of HOXA5-PTD-EGFP were compared by Western blot. EGFP was transformed into Escherichia coli BL21 (DE3) with pET28b / EGFP vector obtained by cleaving the pET28b vector using EcoRI / Xhol restriction enzyme and inserting the EGFP gene sequence amplified in Example 3. Expression and purification were done by the same method. HOXA5-PTD-EGFP and EGFP were each treated with 100 nM concentration in vascular endothelial cells (HUVEC, Human Umbilical Vein Endothelial Cell, American type culture collection) for 1 hour and Western blot analysis. Protein was not introduced into the cell, while HOXA5-PTD-EGFP was confirmed to be introduced into the cell.

Western blot analysis was performed as follows. Cell homogenates were separated by 10% SDS polyacrylamide gel electrophoresis and electrophoresed to the gel with a microcellulose membrane. After the protein-transferred membrane was treated with 5% skim milk for 1 hour, EGFP and β-actin antibody (Santa Cruz Biotechnology), diluted 1: 1000 in 5% skim milk, were reacted at 4 ° C. overnight, followed by TBS-T. Wash three times with buffer for 10 minutes. Secondary antibodies were used as polyclonal anti-rabbit and anti-mouse, and were reacted for 1 hour by diluting 1: 2000 in 5% skim milk. After washing three times with TBS-T buffer three times each 10 minutes and then reacted with ECL reagent (PIERCE Biotechnology) for 1 minute and developed by X-ray film.

(2) Confirmation of concentration dependency of introduction into cells

The HOXA5-PTD-EGFP fusion protein was treated for 1 hour at 1 nM to 200 nM in a concentration-dependent manner for 1 hour after vascular endothelial cells (HUVECs), followed by Western blot in the same manner as in (1). USA). 6 is a graph showing the results, it was confirmed that the introduction of the concentration-dependent introduction into the cell from 30 nM concentration.

(3) Confirmation of time dependence of introduction into cells

100 nM of HOXA5-PTD-EGFP fusion protein was treated with vascular endothelial cells (HUVECs) from 1 nM to 200 nM for 1, 5, 15, 30, 60, 120 minutes and then introduced into cells in the same manner as in (2). The amount of fusion protein was analyzed and the results are shown in FIG. As can be seen in Figure 7, the fusion protein began to flow into the cell from the 5 minutes after the treatment was introduced in a time-dependent manner.

Example 4 Application of HOXA5-PTD to Anti-inflammatory Proteins

1) Preparation of HOXA5-PTD-Ref-1 Expression Vector and Transformant

In order to confirm the applicability of the substantial HOXA5-PTD protein transduction domain, a fusion protein of the anti-inflammatory ref-1 protein and HOXA5-PTD was prepared.

First, human ref-1 cDNA was subjected to polymerase chain reaction from pDsRed-Ref-1 (Jeon et al, Circ Res, 2004) using primers of SEQ ID NOs: 8 and 9 including EcoRI / Xhol restriction enzyme sequences. Isolated (957 bp).

SEQ ID NO: 8 '-cgg aat tcc atg ccg aag cgt ggg aaa aag gga g -3'

SEQ ID NO: 5'- cgc aag ctt tca cag tgc tag gta tag ggt a-'3 '

The EcoOX, XhoI restriction enzyme was used to cleave the pHOXJOIN-N1 vector or pHOXJOIN-C1 vector obtained in Example 2, and then insert the Ref-1 insertion sequence obtained above to insert pET28b / HOXA5-PTD-Ref-1 and pET21b / HOXA5 -PTD-Ref-1 expression vector was completed. A cleavage map of the expression vector is shown in FIG. 8.

Ref-1 expression vector pET28b / Ref-1 was constructed by cutting the pET28b vector with EcoRI / Xhol restriction enzyme and inserting the Ref-1 gene sequence prepared above.

E. coli transformants of each expression vector were prepared by transforming the expression vectors prepared by the above method into E. coli BL21 (DE3). Using the transformant, HOXA5-PTD-Ref-1 fusion protein and Ref-1 were isolated and purified by the same method as Example 3, and used in the next experiment. In the following, only the results of experiments using HOXA5-PTD-Ref-1 expressed from an E. coli transformant of pET21b / HOXA5-PTD-Ref-1 are described, but from the transformant of pET28b / HOXA5-PTD-Ref-1, The expressed HOXA5-PTD-EGFP also showed the same result.

2) Anti-inflammatory Effects of HOXA5-PTD-Ref-1 Fusion Proteins

HUVEC was pretreated with HOXA5-PTD-Ref-1 or Ref-1 at 0, 3, 30, and 100 nM concentrations for 1 hour and then treated with hTNF-α (15 ng / ml) for 12 hours to induce inflammation and reduce monocyte junctions. Monocyte adhesion assays (Song, YJ, et al. (2008), Kim CS et al (2006)) were performed. 9 is a graph showing the results confirmed by the Western blot using a densitometer. As shown in FIG. 9, the Ref-1 treatment group had little inhibitory effect on the inflammatory response, whereas the HOXA5-PTD-Ref-1 fusion protein significantly inhibited the inflammatory response in a concentration-dependent manner.

Figure 1 is an electrophoretic picture of the pHOXJOIN-N1 vector and pHOXJOIN-C1 vector.

2 is a cleavage map of the pHOXJOIN-N1 vector.

3 is a cleavage map of the pHOXJOIN-C1 vector.

Figure 4 is an electrophoresis picture showing the purification and Western blot results of the HOXA5-PTD-EGFP fusion protein.

5 is a photograph and graph of Westton blot results showing the introduction of HOXA5-PTD-EGFP fusion protein into cells.

6 is a graph showing the concentration-dependent introduction of HOXA5-PTD-EGFP fusion protein into cells.

7 is a graph showing that HOXA5-PTD-EGFP fusion protein is introduced into cells in a treatment time dependent manner.

8 is a cleavage map of the pET28b / HOXA5-PTD-Ref-1 vector and the pET21b / HOXA5-PTD-Ref-1 vector.

9 is a graph showing a comparison of the inhibitory effect of HOXA5-PTD-Ref-1 fusion protein and Ref-1.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Transmembrane delivery peptide originated from human homeodomain          gene and protein transduction vector <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> PRT <213> protein transduction domain (PTD) originated from HOXA5, HOXA5-PTD <400> 1 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys   1 5 10 15 <210> 2 <211> 48 <212> DNA <213> gene coding of HOXA5-PTD <400> 2 agacaaatta aaatctggtt ccaaaaccgg agaatgaagt ggaaaaaa 48 <210> 3 <211> 15 <212> PRT <213> PTD originated from Fushi tarazu <400> 3 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Ser Lys   1 5 10 15 <210> 4 <211> 56 <212> DNA <213> oligomer <400> 4 atatgagaca aattaaaatc tggttccaaa accggagaat gaagtggaaa aaacgg 56 <210> 5 <211> 56 <212> DNA <213> oligomer <400> 5 gatcccgttt tttccacttc attctccggt tttggaacca gattttaatt tgtctc 56 <210> 6 <211> 26 <212> DNA <213> primer for amplification of EGFP <400> 6 ggaattcgat ggtgagcaag ggcgag 26 <210> 7 <211> 24 <212> DNA <213> primer for amplification of EGFP <400> 7 ccgctcgagc ttgtacagct cgtc 24 <210> 8 <211> 34 <212> DNA <213> primer for amplification of ref-1 <400> 8 cggaattcca tgccgaagcg tgggaaaaag ggag 34 <210> 9 <211> 31 <212> DNA <213> primer for amplification of ref-1 <400> 9 cgcaagcttt cacagtgcta ggtatagggt a 31  

Claims (8)

A protein transduction domain having the amino acid sequence set forth in SEQ ID NO: 1 from human homeo protein HOXA5. The gene encoding the protein introduction domain of claim 1. The method of claim 2, Gene having the nucleotide sequence of SEQ ID NO: 2. Recombinant vector comprising the gene of claim 2. The method of claim 4, wherein Recombinant vector, characterized in that the pHOXJOIN-N1 or pHOXJOIN-C1 vector having the gene of claim 2 inserted into the EcoRI, XhoI restriction enzyme sequence position of the pET28b vector or pET21b vector. A transformant transformed by the recombinant vector of claim 4. The method of claim 4, wherein Recombinant vector, characterized in that the gene encoding the target protein is inserted together in the vector to express the fusion protein of the target protein and the protein introduction domain of claim 1. A transformant transformed by the recombinant vector of claim 7.

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