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

• Published in: Journal of microbiology and biotechnology Vol. 26; no. 10; pp. 1808 - 1816
• Main Authors: Baek, Jong-Hyuk, Jung, Juneyoung, Seo, Jeongbin, Kim, Jeong Hee, Kim, Joungmok
• Format: Journal Article
• Language: English
• Published: Korea (South) 한국미생물·생명공학회 28.10.2016
• Subjects: Animals; Beclin-1 - chemistry; Beclin-1 - genetics; Beclin-1 - isolation & purification; Beclin-1 - metabolism; Binding Sites - genetics; Class III Phosphatidylinositol 3-Kinases - analysis; Class III Phosphatidylinositol 3-Kinases - metabolism; Escherichia coli - genetics; Mice; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; 생물학; renaturation; Autophagy; Beclin 1; VPS34 binding assay; VPS34-binding domain; denaturing purification
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1604.04085

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 . The expression and purification conditions determined in this study successfully provided soluble and functional Vps34-BD.