title: Bacterial Overexpression and Denaturing Purification of VPS34-Binding Domain of Beclin 1.
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 inEscherichia coli. The expression and purification conditions determined in this study successfully provided soluble and functional Vps34-BD.
authors.affiliation: Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea.
authors.forename: Jong-Hyuk
authors.initials: JH
authors.lastname: Baek
authors.affiliation: Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea.
authors.forename: Juneyoung
authors.initials: J
authors.lastname: Jung
authors.affiliation: Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea.
authors.forename: Jeongbin
authors.initials: J
authors.lastname: Seo
authors.affiliation: Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea.|Department of Oral Biochemistry and Molecular biology, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea.
authors.forename: Jeong Hee
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Oral Biochemistry and Molecular biology, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea.
authors.forename: Joungmok
authors.initials: J
authors.lastname: Kim
mesh_terms: D000818:Animals; D000071186:Beclin-1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D058546:Class III Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000071186:Beclin-1; D011994:Recombinant Proteins; D058546:Class III Phosphatidylinositol 3-Kinases
keywords: Autophagy; Beclin 1; VPS34 binding assay; VPS34-binding domain; denaturing purification; renaturation