Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD)

a technology of icp and parkin, which is applied in the field of new protein-based therapeutic agents, can solve the problems that cp-parkin is not clinically applicabl

Inactive Publication Date: 2017-02-02
JO DAEWOONG +1
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Benefits of technology

[0088]Terminal dUTP nick-end labeling (TUNEL) assays are conducted according to the manufacturers' instructions (Roche). Mouse dopaminergic neuronal (CATH.a) cells (ATCC: American Type Culture Collection) are plated (3×104 / well) and pre-treated with 50 •M 6-hydroxydopamine (6-OHDA) for 1 h at 37° C. followed by the treatment with 2.5 •M Parkin recombinant proteins for 2.5 h at 37° C., analyzing the changes in cell survival. Human brain tumor (SH-SY5Y) cells (Korea Cell Line Bank) are also cultured, plated (3×104 / well) and pre-treated with 100 •M 6-hydroxydopamine (6-OHDA) for 6 h followed by the treatment with 2.5 •M Parkin recombinant proteins for 2.5 h at 37° C., analyzing the alteration.

Problems solved by technology

However, CP-Parkin was not clinically applicable due to its relatively low solubility and yield.

Method used

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  • Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD)
  • Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD)
  • Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD)

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example 1

Construction of Expression Vectors for Recombinant Proteins

[0078]Our newly developed technology, aMTD-based MITT, has enabled us to improve the method for developing cell-permeable recombinant proteins. The expression vectors were designed for Parkin proteins fused with aMTD321 and solubilization domain A (SDA) or solubilization domain B (SDB). To acquire expression vectors for recombinant proteins, polymerase chain reaction (PCR) had been devised to amplify these recombinant proteins.

[0079]The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea)) was digested on the restriction enzyme site between BamHI (5′) and HindIII (3′) involving 35 cycles of denaturation (95° C.) for 30 seconds, annealing (60° C.) for 30 seconds, and extension (72° C.) for 2 min each. For the last extension cycle, the PCR reactions remained for 5 minutes at 72° C. Then, they were cloned into the site of pET...

example 2

Purification and Preparation of Parkin Recombinant Proteins

[0080]Denatured recombinant proteins were lysed using denature lysis buffer (8 M Urea, 10 mM Tris, 100 mM NaH2PO4) and purified by adding Ni-NTA resin. Resin bound to proteins were washed 3 times with 30 mL of denature washing buffer (8 M Urea, 10 mM Tris, 20 m imidazole, 100 mM NaH2PO4). Proteins were eluted 3 times with 30 mL of denature elution buffer (8 M Urea, 10 mM Tris, 250 mM imidazole). After purification, they was dialyzed twice against a refolding buffer (550 mM Guanidine-HCl, 440 mM L-Arginine, 50 mM Tris, 100 mM NDSB, 150 mM NaCl, 2 mM reduced glutathione and 0.2 mM oxidized glutathione). Finally, they were dialyzed against a physiological buffer such as DMEM at 4° C. until the dialysis was over 300×105 times. Concentration of purified proteins was quantified using Bradford assay according to the manufacturer's instructions. After purification, they were dialyzed against DMEM as indicated above. Finally, SDS-PAG...

example 3

Determination of Solubility / Yield of Parkin Recombinant Proteins

[0081]The aMTD321-fused Parkin proteins containing SDA or SDB are cloned, expressed, purified, and prepared in a soluble form. Each recombinant protein fused to aMTD and / or SD was determined for their solubility and yield. Solubility was scored on a 5-point scale ranging from highly soluble proteins with little tendency to precipitate (*****) to largely insoluble proteins (*) by measuring their turbidity (A450). Yield (mg / L) in physiological buffer condition of each recombinant protein was also determined. The cell-permeable Parkin recombinant proteins were observed as a single band, where the amount of the final purified protein was 13 mg / L (FIG. 3).

[0082]Recombinant proteins purified under the denatural condition were analyzed on 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue.

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Abstract

Macromolecule intracellular transduction technology based on improved cell-permeable Parkin recombinant protein (iCP-Parkin) has been developed as a protein-based anti-neurodegenerative agent for efficient BBB-penetration to effectively deliver the recombinant protein into the brain. Parkin protein, a dopaminergic neuronal cell death inhibitor, has been fused with a newly developed advanced macromolecule transduction domain (aMTD) and solubilization domain (SD) to increase the solubility/yield and cell-/tissue-permeability of the recombinant protein. In addition, our newly developed aMTD/SD-fused recombinant iCP-Parkin protein has shown BBB-permeability. Both in vitro and in vivo, our iCP-Parkin recombinant protein improved motor skills, a typical phenotype of Parkinson's disease, by increasing dopamine level in the brain by suppressing apoptosis of dopaminergic neuron cells. In conclusion, iCP-Parkin could be applicable in clinical studies as a protein-based anti-neurodegenerative agent to treat Parkinson's disease by protecting dopaminergic neuron cells and regulating the secretion of dopamine.

Description

BACKGROUND[0001]1. Technical Field[0002]The present invention relates to new protein-based therapeutic agents specially targeted for neurodegenerative disorder based on macromolecule intracellular transduction technology (MITT) enabled with newly advanced hydrophobic CPPs providing cell-permeability of macromolecules in vitro and in vivo. The recombinant protein of this invention has new technical advantages as an intracellular protein therapy for the treatment of Parkinson's disease in that it could resolve blood-barrier permeability, tissue-permeability, and bio-transfer function.[0003]2. Background Art[0004]Parkinson's disease is one of leading neurodegenerative disease that occurs by instable generation and secretion of dopamine (1). In patients with Parkinson's disease, there has been damage in dopaminergic neuron in the midbrain; pathological features, such as a formation of lewy body; mobility defect, such as bradykinesia, rest tremor, and rigidity; and non-motor symptoms, su...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/00G01N33/573
CPCC12N9/93G01N2333/9015C12Y603/02019G01N33/573C07K2319/10A61P25/16A61K38/53C07K7/06C12N15/62C12N15/63C07K2319/21C07K2319/00C07K2319/01
Inventor JO, DAEWOONGHA, EUNSINLEE, JIEUNJEON, JEONGMINLEE, KWANGJAE
Owner JO DAEWOONG
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