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Peptide having cell membrane penetrating activity

a cell membrane and peptide technology, applied in the direction of peptide/protein ingredients, antibody medical ingredients, peptide sources, etc., can solve the problems of limited use of in vivo cellular delivery techniques, extremely limited methods for protein delivery, etc., and achieve high efficiency in drug targeting and prominent penetrating efficiency

Inactive Publication Date: 2013-05-23
EWHA UNIV IND COLLABORATION FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0096]The peptide having cell membrane penetrating activity of the present invention has a prominent penetrating efficiency as compared with the activities of prior TAT-derived peptides and so the peptide has applications on intracellular delivery in various research fields as well as on therapeutics of specific diseases where targeting of drugs is required high efficiently. Accordingly, the peptide having cell membrane penetrating activity of the present invention, the transmembrane complex consisting of the peptide combined with a target substance, and the method for delivering a target substance into a cell using the transmembrane complex is very useful as drug delivery systems.

Problems solved by technology

But such methods are of extremely limited usefulness for delivery of proteins.
However, these techniques have, up to date, shown limited usefulness for in vivo cellular delivery.
However, these methods have not proved to be highly reliable or generally useful.
Recently to introduce macromolecules such as a protein into a cell interior, gene therapy becomes in the limelight but this have also problems in that targeting is incorrect.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0103]Mapping of PTD Using Various Deletion Forms of TCTP

[0104]In order to confirm the region of the TCTP acting as PTD, various deletion constructs were prepared and then used in the experiment as follows.

1) Isolation and Purification of Deletion Forms of TCTP

[0105]To overexpress each of those deletion forms of TCTP (FIGS. 1a and 1c), pRSET vector that is capable of tagging 6 histidine was employed. Subcloning with DNA sequences corresponding to each deletion forms of TCTP was performed in the multicloning site of the vector. Then, the recombinant expression vector was introduced into E. coli BL21(DE3)(Novagen) or BL21(DE3)pLysS (Novagen). The expression of the deletion forms of TCTP was induced by IPTG (isopropyl β-D-thiogalactoside) for 3 hours, then the protein was isolated and purified by using Ni column which binds to polyhistidine.

2) Cell Culture and Treatment with the Protein

[0106]BEAS-2B cell was treated with the deletion form of TCTP at the concentration of 15 ug / ml for 1 ...

example 2

Confirmation of Cell Penetrating Efficiency of the Peptide of the Present Invention

[0112]As shown in Example 1, in order to confirm that the N-terminus of TCTP can function as a PTD, the peptides consisting of N-terminus of TCTP were constructed and examined for cell penetrating efficiency.

1) Synthesis of Various Peptides Corresponding N-Terminus Amino Acid of TCTP

[0113]TCTP-derived peptides and control peptide, TAT 48-57 were synthesized as follow.

Sequence ofClassificationamino acidSEQ ID No.Residues of TCTP(1-10)MIIYRDLISH1Residues of TCTP(1-9)MIIYRDLIS2Residues of TCTP(1-8)MIIYRDLI3Residues of TCTP(2-10)IIYRDLISH4Residues of TCTP(1-7)MIIYRDL5Residues of TCTP(1-6)MIIYRD6Residues of TCTP(3-10)IYRDLISH7Control TAT(48-57)GRKKRRQRRR19

[0114]N-terminus of each peptides was labeled with fluorescence dye, rhodamine and C-terminus was protected. Peptide purity (>95%) was determined by HPLC. Syntersis of the peptides was requested to PEPTRON, Inc.

[0115]Negative control was a fluorescence dy...

example 3

Identification of Intracellular Translocation of TCTP-Derived Peptide by Fluorescence Microscope

[0126]The intracellular translocation of the peptide was identified by fluorescence microscope. HeLa cells were treated with TCTP (1-9)(SEQ ID No.: 2) at a concentration of 10 μM and 100 μM by the same method of Example 2-2). A point of difference was that HeLa cells were seeded in 12 well-plate covered a glass since the plastic plate had a property of fluorescence interference. After washing, cells on cover glass attached slide glass were observed (FIG. 4).

[0127]As shown in FIG. 4, the peptide of the present invention was weakly translocated at a low concentration of 10 μM and strongly at a high concentration of 100 μM. It was found that the peptides were distributed widely in cytoplasm and nucleus of the cell.

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Abstract

Provided are transmembrane complexes that contain a protein transduction domain (PTD) from the N-terminus of IgE-dependent histamine-releasing factor (HRF) and a target substance that is to be delivered into a cell. Also provided are nucleic acid molecules encoding the transmembrane complex, and methods of delivering the target substance into a cell interior by contacting the transmembrane complex with a cell. Also provided are transfection kits containing the PTD and the target substance.

Description

RELATED APPLICATIONS[0001]This application is a continuation of co-pending U.S. application Ser. No. 12 / 280,077, filed Nov. 3, 2008, which is the U.S. National Stage application of PCT / KR2007 / 000885, filed Feb. 20, 2007, entitled “PEPTIDE HAVING CELL MEMBRANE PENETRATING ACTIVITY,” which claims priority to Korean Patent Application No. 10-2006-0016156 filed Feb. 20, 2006, to Kyunglim Lee, Moonhee Kim and Miyoung Kim. The subject matter of each of the above-mentioned applications is incorporated by reference in its entirety.TECHNICAL FIELD[0002]The present invention relates to a peptide having cell membrane penetrating activity, a transmembrane carrier comprising the peptide having cell membrane penetrating activity as an effective component, a transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance, a transfection kit comprising the peptide having cell membrane penetrating activity and the target substance, use of th...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/435C07K16/18
CPCC07K7/06C07K14/47C07K7/08C07K16/18C07K14/435A61K47/65A61K47/64A61P43/00C07K7/00A61K48/0008C07K14/46
Inventor LEE, KYUNGLIM
Owner EWHA UNIV IND COLLABORATION FOUND
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