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Novel macromolecule transduction domains and methods for identification and uses thereof

A large molecule, transduction domain technology, applied in the field of new macromolecule transduction domain peptides, can solve the problems of limited ability to cross the cell membrane and nuclear membrane, toxicity, and poorly understood pathways of cellular uptake

Active Publication Date: 2009-12-30
PROCELL THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, currently known delivery systems exhibit limitations due to their lack of potency and / or their toxicity, and their pathways for cellular uptake are poorly understood, which poses a barrier to improving their potency
Additionally, many delivery systems are limited in their ability to cross cell and nuclear membranes
Even if such delivered peptides do cross cell membranes, their effectiveness is often limited by their internal entrapment in endosomes

Method used

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  • Novel macromolecule transduction domains and methods for identification and uses thereof
  • Novel macromolecule transduction domains and methods for identification and uses thereof
  • Novel macromolecule transduction domains and methods for identification and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1 -Recognition of new macromolecule transduction domain peptides

[0129] In order to identify new macromolecular transduction domain (MTD) candidate peptides that can potentially penetrate through the plasma membrane of living cells, several keywords are used, including "hydrophobic region of signal sequence", "hydrophobic region of signal sequence", and "secreted protein". "Signal sequence", "Hydrophobic signal sequence" and "Hydrophobic region of secreted protein" selected secreted proteins with signal sequence-like domains from the PubMed Entrez protein database. As a result, more than 1,500 secreted proteins with signal sequence-like domains were selected.

[0130] All selected secreted proteins with signal sequence-like domains were subjected to hydrophobicity analysis to determine whether they contained a single hydrophobic region at their N-terminus. Subsequently, computer-assisted analysis of chromosome and proteome information, namely the SOSUI system (a...

Embodiment 2

[0134] Example 2 -Expression of recombinant protein fused to MTD

[0135] After confirming the feasibility of the MTD identified in Example 1 above entering cells, enhanced (type) green fluorescent protein (EGFP) was used as the full-length protein carrier molecule. Green fluorescent protein (GFP) is a protein cloned from jellyfish (Aquorea Victoria). GFP is one of the most widely used reporter proteins and produces green light when irradiated with blue or UV light (Inouye et al., FEBS Letters 351(2): 211-14 (1994)). In one embodiment of the present invention, the commercially available GFP expression vector pEGFP-C1 (Clontech) is used. The EGFP protein encoded by pEGFP-C1 is a mutant of wild-type GFP, which has been modified to produce stronger green light. In addition, when a foreign gene is inserted into multiple cloning sites of the pEGFP-C1 vector, the inserted foreign gene is expressed in the form of a recombinant protein together with EGFP.

[0136] In order to construct ...

Embodiment 3

[0153] Example 3 -Inducible expression and purification of recombinant protein fused to MTD

[0154] In order to express the cell-permeable recombinant protein fused to MTD prepared as described in Example 2 above, in BL21(DE3), BL21-Gold(DE3), BL21-CodonPlus(DE3) and BL21-GoldpLysS(DE3) strains, respectively Transfection of an expression vector containing His-MTD-EGFP recombinant protein. The EGFP expression vector containing kFGF4-derived MTD (SEQ ID NO: 387) was used as a positive control, and the EGFP expression vector fused to a non-functional missense peptide (SEQ ID NO: 389) was used as a negative control.

[0155] After transfection, the cells were grown in LB medium containing kanamycin (30μg / mL) at 37°C with vigorous shaking until the optical density was 600 (OD 600 ) Reaches between 0.4 and 0.6. Then, IPTG (isopropyl-β-D-thiogalactoside) was added to the final concentration of 0.6 mM to induce the expression of His-MTD-EGFP recombinant protein. Protein induction lasted...

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Abstract

The present invention discloses novel macromolecule transduction domain (MTD) peptides which facilitate the traverse of a biologically active molecule across the cell membrane. Also disclosed are polynucleotides encoding the MTD peptides, methods of identifying the MTD peptides; methods of genetically engineering a biologically active molecule to have cell permeability by using the MTD peptides, methods of importing a biologically active molecule into a cell by using the MTD peptides, and uses thereof.

Description

Technical field [0001] The present invention relates to a novel macromolecular transduction domain (MTD) peptide that promotes the penetration of biologically active molecules through cell membranes, a polynucleotide encoding the MTD peptide, a method for identifying MTD peptides, and the use of MTD peptide genetic engineering to design cell-permeable biological activities Molecular methods, methods of importing biologically active molecules into cells using MTD peptides, and their uses. Background technique [0002] Due to the existence of the plasma membrane, the intracellular uptake of macromolecules such as DNA, RNA, proteins, oligonucleotides and peptides is still a challenging task because the plasma membrane constitutes the impermeability of this molecule. barrier. Many difficulties have been encountered in delivering such molecules to the desired target, including poor permeability to tissues or cells, and toxicity due to insufficient specificity of targeting specific tis...

Claims

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Application Information

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IPC IPC(8): C07K7/04C07K7/06C07K7/08
CPCC07K14/47C07K14/195A61K47/48246C07K7/06A61K48/0041A61K39/39C07K7/08A61K47/64C07K7/04C12N15/11C07K2319/00
Inventor 赵大雄高在仙金珍淑朴警湄宋珍京林贞姬杜翠娥杜兰凤杨铭三
Owner PROCELL THERAPEUTICS
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