KR101475744B1 - cell-penetrating peptide - Google Patents

Abstract

The present invention relates to a cell-penetrating peptide derived from a silkworm fluid protein, and more particularly to a cell-penetrating peptide derived from a silkworm fluid 30K protein or a complex comprising the same .

Description

Cell-penetrating peptide < RTI ID = 0.0 >

TECHNICAL FIELD The present invention relates to a cell permeable peptide, and more particularly, to a cell-penetrating peptide derived from a silkworm fluid 30K protein or a complex containing the same.

In drug delivery, intracellular delivery of therapeutic molecules is very important. In order for the drug to exert its therapeutic effect in intracellular organelles such as cytoplasm, nucleus, lysosome and mitochondria, they must pass through the cell membrane. However, immense hydrophilic therapeutic agents such as proteins and nucleic acids can not enter the cell.

One solution to this problem is a " peptide-based delivery system " that utilizes peptide sequences that can cross the cytoplasmic membrane. This system was discovered in 1988 from the TransActivator of Transcription (TAT) part of HIV (Human Immunodeficiency Virus) that can pass through the cell membrane. Since then, it has been found in Antennapediahomeodomain, the third helix of pAnt, that can pass through the cell membrane in 1994. Since then, it has been found that some peptides enter cells, and these peptides are called cell-penetrating peptides (CPP).

A common feature of such a cell-penetrating peptide (CPP) is that it contains a large number of basic sequences such as arginine or lysine, and a specific sequence called PTD (protein transduction domain) consisting of 8-16 amino acids with high positivity I have to have.

Cell permeation of cell-penetrating peptide (CPP) is currently under discussion, but it is not related to specific receptors and is independent of the endocytic pathway. It is known.

Intracellular delivery using cell-penetrating peptide (CPP) can be carried out in subcellular regions of various sites, as the peptide sequence can be modified more than cationic lipid or polyethyleneimine (PEI) (The substance to be transported intracellularly).

The silkworm fluid, when added to the medium in the baculovirus expression system, inhibits the apoptosis of the host cell and increases the survival rate of the host cell. The authors confirmed that silkworm fluid suppresses apoptosis induced by viruses and various chemical substances in insect cells as well as mammalian and human cells, and increases cell growth and productivity.

A substance having an effect of specifically suppressing apoptosis in silkworm fluid is a 30K protein, which is a protein group of about 30 kDa. The 30K protein is a group of structurally similar proteins that consist of five sub proteins with similar molecular weight as well as similar amino acid sequences.

In addition, 30Kc19 is the most abundant protein in the 30K protein. 30Kc19 also suppresses the apoptosis of insect cells and mammalian cells. When applied to a CHO cell system producing EPO (erythropoietin), the production of EPO (Sialyltransferase) activity, thereby increasing the content of sialic acid in the glycoprotein and producing a useful protein of high quality.

In general, animal cells have hydrophobic membranes called lipid bilayers, and it is almost impossible for peptides, proteins, nucleotides, and liposomes to permeate through cell membranes and migrate into cells. However, the inventors of the present invention have found that 30Kc19 is an animal cell and has a property of permeating the cell membrane and moving into the cell. In addition, it has been confirmed that it is located inside the cell to inhibit degradation of the activity of the intracellular enzyme and increase the activity of mitochondria to increase the production of ATP.

It is an object of the present invention to find out that a 30K protein of a silkworm fluid protein has a property of migrating into a cell, and to provide a cell-penetrating peptide of a new structure from such a silkworm solution protein.

Disclosure of the Invention In order to solve the above problems, the present invention provides a cell-penetrating peptide comprising a continuous amino acid represented by the following general formula.

B ₁ -B ₂ - ... -B _{n -1} -B _n ( _n 13)

In the above general formula, B ₁ is Val, B ₁₃ is Cys, and the amino acids except B ₁ and B ₁₃ are independently selected from Ala, Asx, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr and Glx.

The amino acid sequences used in the present invention are abbreviated according to the IUPAC-IUB nomenclature as shown in Table 1 below.

name Abbreviation sign
Alanine  Ala A Arginine  Arg R Asparagine  Asn N Aspartic acid  Asp D Cysteine  Cys C Glutamic acid  Glu E Glutamine  Gln Q Glycine  Gly G Histidine  His H Isoleucine  Ile I Leucine  Leu L Lee Sin  Lys K Methionine  Met M Phenylalanine  Phe F Proline  Pro P Serine  Ser S Threonine  Thr T Tryptophan  Trp W Tyrosine  Tyr Y Balin  Val V

In the cell-penetrating peptide of the present invention, n is 13.

In the cell-penetrating peptide of the present invention, B ₂ is characterized by being Val.

In the cell-penetrating peptide of the present invention, B ₃ , B ₈ , B ₉ , and B ₁₂ are all the same peptides.

In the cell-penetrating peptide of the present invention, B ₄ , B ₇ , and B ₁₀ are basic amino acids. As the basic amino acid, all basic amino acids are possible, and in particular, lysine, arginine (Arg), histidine (His) and the like are preferable.

In the cell-penetrating peptide of the present invention, B ₁ , B ₂ , B ₅ , B ₆ , and B ₁₁ are hydrophobic amino acids. As the hydrophobic amino acid, all hydrophobic amino acids are possible, and in particular, alanine (Ala), valine (Val), leucine (Leu), or isoleucine (Ile) are preferable.

In the cell-penetrating peptide of the present invention, the peptide is characterized in that 10 to 20% of the amino acids are cationic. Since the extracellular membrane is highly negatively charged, it is preferable that 10 to 20% of the cell permeation peptide amino acid is a cationic amino acid so that the peptide can easily adhere to the cell membrane.

The cationic amino acid can be selected from lysine, arginine, histidine and ornithine.

In the cell-penetrating peptide of the present invention, it is preferable that B ₆ and B ₇ are cationic amino acids, B ₆ is isoleucine which is a hydrophobic amino acid, B ₇ is a basic amino acid Arginine.

In the cell-penetrating peptide of the present invention, the peptide is characterized by having a Helix-Loop-Helix motif.

The cell-penetrating peptide of the present invention is characterized in that it is derived from a silkworm protein.

The cell-penetrating peptide of the present invention is characterized in that it comprises SEQ ID NOS: 1-4.

SEQ ID NO: 1 - VVNKLIRNNKMNC

SEQ ID NO: 2 - NVVNKLIRNNKMNC

SEQ ID NO: 3 - TNVVNKLIRNNKMNC

SEQ ID NO: 4 - ITNVVNKLIRNNKMNC

In the cell-penetrating peptide of the present invention, it is possible that cargo of an intracellular carrier is additionally bound to the terminal of the peptide.

In the cell-penetrating peptide of the present invention, the cargo molecule is covalently or non-covalently bound to the peptide.

In the cell-penetrating peptide of the present invention, the cargo to be transported is characterized by comprising a nucleic acid, an amino acid, a peptide, a polypeptide, a carbohydrate, a lipid and a small molecule, and a mixture of any of them .

The cell-penetrating peptide derived from the silkworm protein according to the present invention has a property of permeating through the cell membrane, thereby effectively transferring the bound foreign substance (cargo molecule) into the cell, Can be used as a tool.

Fig. 1 shows the result of analysis of the secondary structure of 30Kc19 protein using the CLC protein workbench program of Insilicogen.
FIG. 2 shows the result of predicting the surface charge of 30Kc19 protein using the software provided by Pasteur Laboratories.
Figure 3 shows the hydrophobicity of 30Kc19 protein using the CLC protein workbench program of insilicogen.
FIG. 4 is a result of estimating the solvent accessibility of proteins of 30Kc19 using the LOOPP (Program of Observing and Outputting Protein) program of Cornell University Computational Biology Service Unit (CBSU).
FIG. 5 shows fluorescence intensity of a FITC-peptide according to an embodiment of the present invention using a spectrofluorometer.
FIGS. 6 to 11 show the results of a cell permeation reaction of a peptide according to an embodiment of the present invention observed using a confocal microscope.
FIG. 12 is a graph showing fluorescence intensities measured using a spectrofluorometer to measure the intracellular permeability before and after treatment of FITC-peptide according to the present invention with cytochalasin B and sucrose .
Fig. 13 shows the result of confirming the intracellular fluorescence of a cell infiltration pattern of 30Kc19 and 30Kc19 CPP with GFP attached using a fluorescence microscope and a confocal microscope.
Fig. 14 shows the result of confirming intracellular fluorescence using a fluorescent microscope after HeLa cells were treated with the above (GFP-peptide of Example 12) and the protein of the comparative example for 4 hours.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited by the following examples.

< Example > Cell permeation from silkworm fluid protein Peptides  design

In order to isolate the CPP-expressing peptide portion from 30Kc19, a silkworm protein of known sequence, a portion satisfying the following characteristics required for a cell-penetrating peptide was predicted using a computer program.

i) Helix-Loop-Helix motif

ii) Positive surface charge

iii) hydrophobic

iv) Solvent accessibility

The computer program predictions for i) to iv) above are shown in FIGS. 1 to 4, respectively. Seventeen portions of peptides having all of the features of i) to iv) above were designed as shown in Table 2 below.

FITC-30Kc19 peptide Example 1 FITC-VITNVVNKLIRNNKM Example 2 FITC-ITNVVNKLIRNNKM Example 3 FITC-TNVVNKLIRNNKM Example 4 FITC-NVVNKLIRNNKM Example 5 FITC-VVNKLIRNNKM Example 6 FITC-VITNVVNKLIRNNKMN Example 7 FITC-ITNVVNKLIRNNKMN Example 8 FITC-TNVVNKLIRNNKMN Example 9 FITC-NVVNKLIRNNKMN Example 10 FITC-VVNKLIRNNKMN Example 11 FITC-VITNVVNKLIRNNKMNC Example 12 FITC-ITNVVNKLIRNNKMNC Example 13 FITC-TNVVNKLIRNNKMNC Example 14 FITC-NVVNKLIRNNKMNC Example 15 FITC-VVNKLIRNNKMNC Example 16 FITC-VNKLIRNNKMNC Example 17 FITC-NKLIRNNKMNC

< Experimental Example  1> Fluorescence spectrometer ( 스펙트로fluorometre ) Of peptide  Intracellular permeability measurement

HEK 239 or HeLa was used as a cell line for the experiment of permeation of 17 peptides designed in the above example into cells.

HEK 293 and HeLa were cultured in an incubator containing 5% CO ₂ at 37 ° C using DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (fetal bovine serum) Was prepared, and the cells were cultured at 37 캜.

The permeability of the 17 peptides designed in the above example into HEK 239 cells was measured using a spectrofluorometer and the results are shown in FIG. In FIG. 5, the peptides of Examples 1, 2, 6, 7, 14 and 15 have excellent intracellular permeability.

< Experimental Example  2> Confocal microscopy  Used Of peptide  Intracellular permeability measurement

Among the 17 FITC-peptides of Examples 1 to 17, six examples (Examples 1, 2, 6, and 7) in which the degree of intracellular penetration of the FITC-peptide using the fluorescence spectrometer of Experimental Example 1 was high , 14, 15) FITC-peptide was selected.

The FITC-peptides of the six examples thus selected were measured for the degree of fluorescence inside the cells using a confocal microscope.

The FITC-peptide was added to the HeLa cells at a concentration of 10 mM, and the cells were incubated for a predetermined time and washed three times with PBS. Hoechst 33342 for staining the nuclei of the washed cells was treated for 10 minutes, and cells were observed using a confocal microscope (Nikon, Japan). The results are shown in FIGS. 6 to 11. As shown in FIGS. 6 to 11, in Examples 1, 2, 6, and 7, no cell permeation reaction was observed, and peptides were observed outside the cells. In Examples 14 and 15, The fluorescent material was also detected.

< Experimental Example  3> Of peptide  Measurement of intracellular permeation mechanism

In order to examine the intracellular permeation mechanism of the peptides of Examples 14 and 15, which were measured with high transmittance in Experimental Example 2, intracellular permeability before and after treatment with cytokalase B and sucrose was measured using a fluorescence spectrometer And the results are shown in Fig.

In FIG. 12, peptides of Examples 14 and 15 showed intracellular penetration, microfilament collapse and macropinocytosis, although the degree of intracellular permeability was decreased when treated with cytokalase B and sucrose. 15 peptides, and that clathrin-mediated cytotoxins are involved in the cell infiltration of the peptides of Examples 14 and 15.

< Experimental Example  4> GFP -30 Kc19 CPP  Fusion protein production and cell permeation effect analysis

In general, cargo, which is a large substance which can not be transferred into the cell, is bound to analyze whether the peptide of the present invention can transfer an external large molecule into a cell.

In this Experimental Example, fluorescently-labeled GFP was selected as a cargo-exogenous protein, and as a peptide, in Example 12 in which amino acid was further bonded to the end of the peptide of Example 14 showing the cell penetration effect in Experimental Example 3 A peptide (GFP-peptide of Example 12), in which GFP and a peptide were fluorescently attached as an exogenous protein, was prepared as follows.

The gene of the peptide of Example 12 was inserted into a pET23a vector (Novagen) at EcoRI and XhoI sites and ligated to obtain a vector.

The GFP gene was inserted into the BamH I and EcoR I sites of the vector and ligation was performed to complete the plasmid for producing the fusion protein. The E. coli strain BL21 (DE3) transformed with the above plasmid was cultured in LB medium, and 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added And then cultured at 30 캜 for 6 hours to produce a GFP-peptide fusion protein in which GFP was bound to the N-terminus of the gene of the peptide of Example 12. Thereafter, the cells collected through centrifugation were sonicated. At this time, the produced fusion protein was produced by binding the peptide of Example 12 to the carboxyl terminal of GFP and binding a histidine tag to the carboxyl terminal of the peptide to facilitate purification.

The supernatant obtained by centrifuging the above-mentioned disruption solution was purified by using HisTrap HP column (GE Health care) (GFP-peptide of Example 12). The purified (GFP-peptide of Example 12) was buffer exchanged with Tris-HCl (pH 8), stored and used in the experiment.

As a comparative example, 30Kc19 whole protein, GFP, and GFP-30Kc19 whole protein were used. (GFP-30Kc19 whole protein) was prepared in the same manner as above except that 30Kc19 protein was used instead of the peptide. Fig. 13 shows the structure of the protein used in the experiment.

The result of treating the above (GFP-peptide of Example 12) and the protein of Comparative Example for 4 hours in HeLa cells and confirming the fluorescence in the cells using a fluorescence microscope is shown in Fig. In Fig. 14 (GFP-peptide of Example 12), it can be confirmed that the peptide is permeated into the cell.

<110> seoul national university <120> cell penetrating peptide <130> DPP-2012-0038 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 1 Val Val Asn Lys Leu Ile Arg Asn Asn Lys Met Asn Cys   1 5 10 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 2 Asn Val Val Asn Lys Leu Ile Arg Asn Asn Lys Met Asn Cys   1 5 10 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 3 Thr Asn Val Val Asn Lys Leu Ile Arg Asn Asn Lys Met Asn Cys   1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 4 Ile Thr Asn Val Val Asn Lys Leu Ile Arg Asn Asn Lys Met Asn Cys   1 5 10 15

Claims (15)

A cell-penetrating peptide comprising one peptide selected from the four peptides consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 4.
delete delete delete delete delete delete delete delete The method according to claim 1,
Wherein the peptide has a Helix-Loop-Helix motif.
The method according to claim 1,
Wherein the peptide is derived from a silkworm protein.
delete The method according to claim 1,
Wherein the cargo of the intracellular carrier is additionally bound to the end of the peptide.
14. The method of claim 13,
Wherein the cargo is covalently or non-covalently bound to the peptide.
14. The method of claim 13,
Wherein the cargo comprises a nucleic acid, an amino acid, a peptide, a polypeptide, a carbohydrate, a lipid and a small molecule, and any mixture thereof.

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