CELL-PENETRATING PEPTIDE DIMERS, METHOD FOR PREPARING THE SAME, AND CARGO DELIVERY SYSTEM USING THE SAME

Abstract

The present invention relates to a cell-penetrating peptide dimer comprising: a first peptide domain consisting of the amino acid sequence of SEQ ID NO: 1; a second 30Kc19α peptide domain consisting of the amino acid sequence of SEQ ID NO: 1; and a peptide linker connecting the first and second peptide domains, a method for preparing the peptide dimer, a cargo delivery system in which a cargo is conjugated to the dimer; and a use thereof. The cell-penetrating peptide dimer according to the present invention may have excellent cell-penetrating properties, thereby being usefully employed as the cargo delivery system.

Claims

1. A cell-penetrating peptide dimer comprising: a first peptide domain consisting of the amino acid sequence of SEQ ID NO: 1; a second peptide domain consisting of the amino acid sequence of SEQ ID NO: 1; and a peptide linker connecting the first and second peptide domains, wherein the cell-penetrating peptide dimer is a fusion protein, and the peptide linker connects a carboxyl-terminus of the first peptide domain and an amino-terminus of the second peptide domain to each other, and consists of 100 or less amino acids, wherein the peptide linker is a flexible linker or a rigid linker.

2. The cell-penetrating peptide dimer according to claim 1, wherein the peptide linker is the flexible linker including glycine (G) and serine (S).

3. The cell-penetrating peptide dimer according to claim 2, wherein the peptide linker is the flexible linker comprising an amino acid sequence repeating unit consisting of glycine (G) and serine (S).

4. The cell-penetrating peptide dimer according to claim 1, wherein the peptide linker is a rigid linker comprising repeating units of amino acid sequence EAAAK.

5. The cell-penetrating peptide dimer according to claim 1, wherein the peptide linker consists of the amino acid sequence of any one of SEQ ID NOs: 2 to 8.

6. The cell-penetrating peptide dimer according to claim 1, wherein a cargo is conjugatable.

7. A nucleic acid encoding the cell-penetrating peptide dimer according to claim 1.

8. The nucleic acid according to claim 7, wherein the nucleic acid encoding the peptide linker connects the 3′end of the nucleic acid encoding the first peptide domain and the 5′ end of the nucleic acid encoding the second peptide domain to each other.

9. The nucleic acid according to claim 8, which further comprises a nucleic acid encoding a cargo that is a peptide or a protein, wherein the nucleic acid encoding the cargo is connected to the 5′end of the nucleic acid encoding the first peptide domain or the 3′ end of the nucleic acid encoding the second peptide domain.

10. A cargo delivery system comprising the cell-penetrating peptide dimer according to claim 1 and a cargo conjugated to the dimer.

11. The cargo delivery system according to claim 10, wherein the cargo is a peptide, a protein, a nucleic acid, a lipid, a glycolipid, a carbohydrate, a mineral, a nanoparticle, a virus, a contrast material, a chemical substance, or a combination thereof.

12. The cargo delivery system according to claim 11, wherein the cargo is covalently linked to the cell-penetrating peptide dimer.

13. The cargo delivery system according to claim 12, wherein the cargo is covalently linked to an amino-terminus of the first peptide domain or a carboxyl-terminus of the second peptide domain of the cell-penetrating peptide dimer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1 shows a cell-penetrating peptide dimer of the present invention;

[0071] FIGS. 2A, 2B, 3A, 3B, 4A, and 4B visually explain the improved cell-penetration effect of the cell-penetrating peptide dimer of the present invention;

[0072] FIG. 5 is an image showing results of preparing the cell-penetrating peptide dimer of the present invention;

[0073] FIG. 6 is an image confirming an intracellular cell-penetration ability of the cargo-conjugated cell-penetrating peptide dimer of the present invention by immunocytochemistry; and

[0074] FIG. 7 shows a graph showing comparison of cell-penetration abilities of the cargo-conjugated cell-penetrating peptide dimers of the present invention using various linkers.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0075] Hereinafter, the present invention will be described in more detail by Examples. However, these Examples of the present invention are provided to facilitate understanding of the invention, and the scope of the invention to be protected is not limited by the following Examples.

[0076] <Experimental Materials and Methods>

[0077] 1. Construction of Plasmid

[0078] As a plasmid for production of a recombinant protein, a pET-23a vector (Novagen, Madison, Wis., USA), which is advantageous for expression in E. coli and purification of His tag protein, was purchased and used. The entire 30Kc19α genes containing a linker or 30Kc19α were synthesized by Geneart. During the synthesis, codon optimization was performed according to the E. coli strain.

[0079] The TurboGFP-30Kc19α gene sequence (SEQ ID NO: 10) in which the TurboGFP gene sequence was fused to the upstream of the 30Kc19α gene sequence (SEQ ID NO: 9) was inserted using the BamHI/Xhol restriction enzyme site in the multiple cloning site (MCS) of the pET-23a vector. The synthesized genes were inserted into the pET-23a vector containing TurboGFP using the EcoRI/Xhol restriction enzyme site in the sequence.

[0080] Specifically, first, each vector and insert added with EcoRI/Xhol enzyme (NEB) and a custom buffer were cultured for 18 hours in a heat block at 37° C. Next, the insert and vector were subjected to electrophoresis on an agarose gel, and then each insert and vector were extracted from the gel using AccuPrep® PCR/Gel DNA Purification Kit (Bioneer). The extracted insert and vector were mixed in a ratio of 3:1, and then T4 DNA ligase (Cat No. M0202M from NEB) was added and cultured for 18 hours. The resulting plasmid was transformed into DH5a competent cells (Cat. No. RH617-J80 from RBC), and then only ampicillin resistant colonies were selected and cultured in 2 ml of LB broth media (0.1% ampicillin) for 12 hours. The plasmid was extracted from the cell pellets using AccuPrep® Nano-Plus Plasmid Extraction Kit (Bioneer), and the final nucleotide sequence was confirmed.

[0081] 2. Preparation and Purification of Recombinant Protein

[0082] The plasmid extracted according to the above-described procedure was transformed into BL21 competent cells, put into LB broth medium, and cultured in a shaking incubator. The product was treated with IPTG 1 mM, and cultured for 4 hours. Cell lysates were obtained using a centrifuge and lysed using a sonicator. Then, the protein was purified using FPLC (GE Healthcare), followed by dialysis, and stored. As purification buffers, lysis buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 8.0), washing buffer (20 mM Tris-HCl, 0.5 M NaCl, 50 mM imidazole, pH 8.0), elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 350 mM imidazole, pH 8.0), and dialysis buffer (20 mM Tris-HCl buffer, pH 8.0) were used.

[0083] As a result, recombinant proteins (Examples 1 to 7) having the structure of N′-GFP-30Kc19α-linker-30Kc19α-C′ were obtained. All of the GFP-30Kc19α contained in the recombinant proteins of Examples 1 to 7 had the amino acid sequence of SEQ ID NO: 1, and the linkers had the amino acid sequences shown in the following table. Here, the linkers L5, L10, L20, L30, L40 and L50 were flexible linkers having 5 to 50 amino acids in length (GGSSS repeated sequence), and Rigid 20 is a rigid linker having 20 amino acids in length (EAAAK repeated sequence).

TABLE-US-00002 Example Linker Amino Acid Sequence SEQ ID NO. Example 1 L5 GGSSS SEQ ID NO. 2 Example 2 L10 GGSSSGGSSS SEQ ID NO. 3 Example 3 L20 GGSSSGGSSSGGSSSGGSSS SEQ ID NO. 4 Example 4 L30 GGSSSGGSSSGGSSSGGSSSGGSSSGGSSS SEQ ID NO. 5 Example 5 L40 GGSSSGGSSSGGSSSGGSSSGGSSSGGSSS SEQ ID NO. 6 GGSSSGGSSS Example 6 L50 GGSSSGGSSSGGSSSGGSSSGGSSSGGSSS SEQ ID NO. 7 GGSSSGGSSSGGSSSGGSSS Example 7 Rigid 20 EAAAKEAAAKEAAAKEAAAK SEQ ID NO. 8

[0084] As Comparative Peptides, a recombinant protein having a structure of N′-GFP-30Kc19α-C′ (Comparative Example 1) and a recombinant protein having a structure of N′-GFP-30Kc19α-30Kc19α-C′ (Comparative Example 2) were also prepared. The GFP-30Kc19α contained in Comparative Examples 1 and 2 all had the amino acid sequence of SEQ ID NO: 1.

[0085] The prepared protein was confirmed using SDS-PAGE. The prepared protein having an amount of 3 μg was mixed with 4× Laemmli sample buffer (Bio-Rad, Cat. No. 1610747) and heated for 5 minutes using a 95° C. heat block. Each sample was loaded onto a 4-15% Mini-PROTEAN TGX stain-free gel (Bio-Rad, Cat. No. 456-1085), then stained with Coomassie Brilliant Blue G-250 (ThermoFisher Scientific, Cat. No. 20279), and observed. Results of the prepared proteins were shown in FIG. 5.

EXPERIMENTAL EXAMPLE

Experimental Example 1: Confirmation of Cell-Penetration Ability of Cell-Penetrating Peptide Dimer Using Immunocytochemistry

[0086] For immunocytochemistry experiments, HeLa cells and the GFP-conjugated cell-penetrating peptide dimer (Example 2) according to the present invention were incubated for 4 hours, and then strongly washed three times with PBS. The cells were fixed by treatment with 4% paraformaldehyde for 20 minutes, followed by incubation with PBS containing 0.25% Triton X-100 for 10 minutes to perform permeabilization. The fixed cells were blocked with 0.1% PBS-T containing 3% BSA for 1 hour. Then, the cells were incubated at 4 t for 16 hours with an anti-Rab-7 antibody (anti-mouse) (Cat. No. ab50533; Abcam, USA) and an anti-turboGFP antibody (anti-rabbit) (Invitrogen, Cat. No. PAS-22688). After washing three times with PBS for 10 minutes each, the cells were treated for 1 hour with secondary antibodies, that is, an anti-rabbit antibody (Invitrogen, Cat. No. A32731) and an anti-mouse antibody (Invitrogen, Cat. No. A32744) each diluted to 1:2000. After washing again 5 times with PBS for 10 minutes each, the cell nuclei were stained with Hoechst 33342 for 10 minutes. After washing with PBS for 10 minutes, intracellular fluorescence was observed using a confocal laser microscope (Leica, Germany), and images were taken using software (Leica). Results are shown in FIG. 6.

[0087] In FIG. 6, blue fluorescence indicates cell nuclei, red fluorescence indicates intracellular distribution of the endosome marker Rab7, and green fluorescence indicates intracellular distribution of GFP. In the control group to which the peptide was not added to the HeLa cells (Control) and the experimental group treated with the GFP protein alone (GFP), the green fluorescence did not show in the cells, whereas, in the experimental group treated with the GFP-conjugated cell-penetrating peptide dimer (Example 2), the green fluorescence was clearly observed in the cells. That is, it was confirmed that the cell-penetrating peptide dimer according to the present invention had an effective cell-penetration ability (FIG. 6).

Experimental Example 2: Confirmation of Cell-Penetration Ability of Cell-Penetrating Peptide Dimers According to Various Linkers

[0088] HeLa cells were seeded in a 96-well plate to have a confluency of 70% and stabilized for 24 hours. The GFP-conjugated peptides of Examples 1 to 7 prepared in the above-described Examples were added to the medium at a final concentration of 1 μM. After 1 hour, the peptides were washed three times with PBS, and the GFP fluorescence intensity was measured using a plate reader.

[0089] As a result, it was confirmed that the cell-penetrating peptide dimer according to the present invention had significantly improved cell-penetrating properties compared with those of not only the peptide of Comparative Example 1, which was the cell-penetrating peptide monomer but also the peptide of Comparative Example 2, which was the peptide dimer without using the linker. Furthermore, it was confirmed that all of the cell-penetrating peptide dimers of the present invention using linkers having various lengths and amino acid configurations had remarkably excellent cell-penetrating properties compared with those of Comparative Examples (FIG. 7).

[0090] The cell-penetrating peptide dimer according to the present invention has remarkably excellent cell-penetrating properties compared with those of the 30Kc19α peptide monomer, thereby being usefully employed as a cargo delivery system.

[0091] Although some Examples of the present invention have been shown and described, those skilled in the art having ordinary skill in the art to which the present invention pertains will appreciate that the present embodiments can be modified without departing from the principles or spirit of the present invention. The scope of the present invention will be determined by the appended claims and equivalents thereof.