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KMID : 0545120160260101808
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Journal of Microbiology and Biotechnology
2016 Volume.26 No. 10 p.1808 ~ p.1816
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Bacterial Overexpression and Denaturing Purification of VPS34-Binding Domain of Beclin 1
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Baek Jong-Hyuk
Jung June-Young
Seo Jeong-Bin
Kim Jeong-Hee
Kim Joung-Mok
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Abstract
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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.
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KEYWORD
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Autophagy, Beclin 1, VPS34-binding domain, denaturing purification, renaturation, VPS34 binding assay
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