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已有 5751 次阅读 2010-7-7 08:33 |个人分类:未分类|系统分类:科研笔记

Title:The over expression and purification of a apoptotic suppressor-Bcl-xL and its BH3 domain
Abstract
Apoptosis is a morphologically distinct form of programmed cell death essential for normal development and tissue homeostasis. The BCL-2 family of proteins constitutes a critical control point in apoptosis residing immediately upstream of irreversible cellular damage.
Bcl-xL plays a critical role in maintaining cell survival. One group has identified A protein , a multifunctional protein, as a novel antiapoptotic Bcl-xL-interacting protein. However, in fact, another previous study did not identify the interaction between A and Bcl-xL. We cloned the Bcl-xL and its BH3 domain from human into a pET28-derivative vector, then produced the protein in E. coli Rossetta(DE3) cells. We harvested the cells by centrifugation after 3 h induction with 1 mM ITPG when the optical density at 600 nm reached 0.8, and resuspended the pellet in ice-cold Tris buffer and froze the cells at 253 K. The frozen cells were thawed and lysed by sonication. We centrifuged the crude extract at 16000 rpm for 30 min and the supernatants were applied on a metal ion affinity chromatography column. The elutions by 300 mM imidazole were dialysed and digested with PPase to remove the fusion tag. The protein Bcl-xL and BH3 pepites were further purified by passing through a metal ion affinity chromatography column,and by gel filtration on a Sephadex G-75 column. The purity was detected by SDS-PAGE and Trincine-SDS-PAGE, which indicated that the purified protein and peptide were suitable for NMR study.
Keywords: Apoptosis Bcl-xL BH3 expression purification


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