Expression, purification, and in vitro mitochondrial interaction analysis of full-length and truncated human tumor suppresser p53. Issue 7 (3rd July 2019)

• Record Type: Journal Article
• Authors: Wang, Lingyao; Zhao, Tong; Wang, Song; Jin, Jingji; Cai, Yong; Wang, Fei
• Is Part Of: Bioscience, biotechnology, and biochemistry. Volume 83:Issue 7(2019)
• Page Start: 1220
• Page End: 1226
• Publication Date: 2019-07-03
• Subjects: P53 -- truncation -- His-tag fusion protein -- soluble expression -- cell-free apoptosis analysis; Biotechnology -- Periodicals; Biochemistry -- Periodicals; 660.6
• DOI: 10.1080/09168451.2019.1594674
• Languages: English
• ISSNs: 0916-8451

Abstract

ABSTRACT: p53 is a potent tumor suppressor which can prevent the propagation of cells carrying oncogenic lesions via a multitude of pathways. Besides the transactivation of downstream genes encoding proapoptotic proteins, p53 is also able to physically interact with mitochondria and induce apoptosis through a so called transcriptional-independent pathway. In this study, we described a quick method for the expression and purification of soluble recombinant p53 and its different truncations in E. coli . These proteins are able to interact with mitochondria and induce mitochondrial outer membrane permeabilization and associated downstream apoptotic events in a cell-free apoptosis analysis system. Graphical abstract: : DNA binding domain of p53 is essential for its direct interaction with isolated mitochondria in vitro . Abbreviations : MOMP: mitochondrial outer membrane permeabilization; DBD: DNA binding domain; PCR: polymerase chain reaction; LB medium: Luria Bertani medium; IPTG: isopropyl β-d-thiogalactoside; Ni 2+ -NTA resin: nickel-nitrilotriacetic acid resin; PVDF membrane: polyvinylidene difluoride membrane;