Preparation and Characterization of the Self-releasing Intracellular Transporter LCA2 Domain
LIU Di 1)*, WU Xiao-Ying1), WANG Shu-Hui2), QIAO Hong-Ping1), LI Na1), CHAI Mei-Ling1), MA Xing-Yuan3)*
1)Department of Biology, Center for Veterinary Medicine, Taiyuan Normal University, Jinzhong 030619, Shanxi, China; 2)Department of Animal Production, College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi, China; 3)School of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
Abstract：Protein drugs play an extremely important role in the prevention and treatment of diseases. But the properties of macromolecules hinder their effects on intracellular targets. Among the existing delivery strategies, penetrating peptides are more suitable for clinical research and treatment, and have gradually become the most important tool to deliver protein drugs. Therefore, the development of safe and effective penetrating peptide delivery vehicles is of great significance to the basic research and clinical application of biomedicine. In this paper, a self-releasing intracellular transporter LCA2 based on the enterotoxin A2 domain is designed. This carrier is composed of three parts: a linker, self-releasing enzyme sensitive sites (Cs), and the transmembrane domain LTA2. The fluorescent protein mCherry was used as the model protein to detect the properties of LCA2. The results of electrophoresis showed that the high-purity mCherry-LCA2 fusion protein was obtained from the engineered bacteria containing pET24a(+)-ma2 recombinant plasmids, and mCherry could be effectively separated from LCA2 by low concentration trypsin. It was observed under a fluorescence microscope that LCA2 could transport mCherry into different types of cells. Flow cytometry has detected that the transport capacity of LCA2 has certain cellular differences. Confocal microscope fluorescence analysis and Western blotting results showed that the mCherry was transported to the endoplasmic reticulum by the LCA2 carrier, separated from LCA2 by cleavage of enzyme sensitive sites and released into the cell. The CCK-8 results showed that there was no significant change in cell viability within the dose range of 5-40 μg/mL. These results demonstrate that LCA2 is a safe and effective self-releasing delivery vehicle, which can transport and release active proteins or protein drugs into cells.
刘地, 武晓英, 王姝慧, 乔宏萍, 李娜, 柴美灵, 马兴元. 自释放型细胞内运输载体LCA2结构域的制备与性质研究[J]. 中国生物化学与分子生物学报, 2021, 37(10): 1366-1376.
LIU Di, WU Xiao-Ying , WANG Shu-Hui , QIAO Hong-Ping , LI Na , CHAI Mei-Ling , MA Xing-Yuan. Preparation and Characterization of the Self-releasing Intracellular Transporter LCA2 Domain. Chinese Journal of Biochemistry and Molecular Biol, 2021, 37(10): 1366-1376.
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