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http://dx.doi.org/10.4014/jmb.1604.04085

Bacterial Overexpression and Denaturing Purification of VPS34-Binding Domain of Beclin 1  

Baek, Jong-Hyuk (Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University)
Jung, Juneyoung (Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University)
Seo, Jeongbin (Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University)
Kim, Jeong Hee (Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University)
Kim, Joungmok (Department of Oral Biochemistry and Molecular biology, School of Dentistry, Kyung Hee University)
Publication Information
Journal of Microbiology and Biotechnology / v.26, no.10, 2016 , pp. 1808-1816 More about this Journal
Abstract
As a scaffolding subunit of the PIK3C3/VPS34 complex, Beclin 1 recruits a variety of proteins to class III phosphatidylinositol-3-kinase (VPS34), resulting in the formation of a distinct PIK3C3/VPS34 complex with a specific function. Therefore, the investigation of a number of Beclin 1 domains required for the protein-protein interactions will provide important clues to understand the PIK3C3/VPS34 complex, of which Beclin1-VPS34 interaction is the core unit. In the present study, we have designed a bacterial overexpression system for the Beclin 1 domain corresponding to VPS34 binding (Vps34-BD) and set up the denaturing purification protocol due to the massive aggregation of Vps34-BD in Escherichia coli. The expression and purification conditions determined in this study successfully provided soluble and functional Vps34-BD.
Keywords
Autophagy; Beclin 1; VPS34-binding domain; denaturing purification; renaturation; VPS34 binding assay;
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