TEST RESULTS

SNAP25 Cleavage Diagram

Recombinant GST-SNAP25-EGFP is purified in order to review activation of 'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40' protein. Since wild type Botulinum Toxin Light Chain cleaves an  C-terminal part of SNAP25, GST & EGFP are recombined with 25kDa size on each N-terminal and C-terminal, and purified in order to authentically confirm cleavage form. 75kDa GST-SNAP25-EGFP is cleaved into 50kDa and 25kDa proteins by 'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40'.

SNAP25 Cleavage

Size alteration after GST-SNAP25-EGFP cleavage

GST

GST

SNAP25

SNAP25

EGFP

EGFP

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75kDa

50kDa + 25kDa

in-vitro SNAP25 cleavage assay

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GST-SNAP25-EGFP

GST-SNAP25

EGFP

Buffer condition: 1mM DTT, 1mM HEPES, 1mM NaCl, 2uM ZnCl₂

Reaction : 37℃ 3hrs

SDS-PAGE loading condition

- 12% SDS-PAGE

- volt : 120V

in vitro SNAP25 Cleavage Assay

React GST-SNAP25-EGFR protein with various dose of  'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40' in vitro to see if GST-SNAP25-EGFP is cleaved by  'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40'. As a result, GST-SNAP25-EGFP protein is cleaved into GST-SNAP25(50kDa) and EGFP(25kDa) depend on dose of  'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40'.

Cell Penetrate Assay

Vehicle

Lc

BPMed_BoLCA

1 hr

3 hr

6 hr

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Permeation of Keratinocytes by ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ 

Permeation of keratinocytes 

Cell treatment on Keratinocytes cell with FITC conjugated ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ protein. After 1~6 hours, protein can be seen inside the cell. This result shows that  ‘Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40’ can penetrate skin.

* Treatment condition

- HaCaT cell

- Peptide : 5μm

Vehicle

Lc

BPMed_BoLCA

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'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40' cell penetrate test

Potential in human neuronal cell

Cell treatment on differentiated SiMa cell (Human neuroblastoma) with FITC conjugated 'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40' protein. After 3 hours, protein can be seen inside the cell. This result shows that  'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40' can be delevered to the human nerve cell, the most important target of Botulinum Protein.

* Treatment condition

- FITC conjugated : 5μg

- Time : 3hrs

- Method : confocal

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'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40' transdermal diffusion test 

Transdermal Diffusion

Perform transdermal diffusion test by using Strat-M membrane which is a product from Millipore. Conversion of  'Methionyl r-Clostridium Botulinum Polypeptide-1 Hexapeptide-40' amount which is penetrated from 30minutes to 24 hours, into cumulative frequency by using ELISA. Penetrated continuously even after 24 hours.