Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo
Author:
Hao Fengjin1, Feng Yueqin2, Guan Yifu1
Affiliation:
1. Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning, 110122, China 2. Department of Ultrasound, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, 110001, China
Abstract
Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal
targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane
biological activity.
Methods:
We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed
and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify
whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25.
Results:
Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence,
and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze
intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition,
the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain
tissue.
Conclusion:
TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins
across the cell membrane without influencing the biological activity.
Publisher
Bentham Science Publishers Ltd.
Subject
Molecular Biology,Biochemistry
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