Purification and characterization of the bcl-2 protein.
Purification and characterization of the bcl-2 protein.
复制标题
bcl-2 蛋白的纯化和表征。
DOI：
10.1006/abbi.1994.1529
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发表时间：
1994
影响因子：
3.9
通讯作者：
Croce,CM
中科院分区：
文献类型：
--
作者：
Haldar,S;Jena,N;DuBois,GC;Takayama,S;Reed,JC;Fu,SS;Croce,CM
癌基因产物bcl-2作为程序性细胞死亡的阻遏物起作用，并且是一种26-kDa的蛋白质，具有位于羧基末端的单个预测跨膜区段。bcl-2蛋白似乎在不同的亚细胞区室中起作用，这一点已被一些生化和超微结构研究所证实。本研究进行纯化bcl-2蛋白的结构和功能研究所需的显着数量。为此，bcl-2基因在杆状病毒系统或淋巴细胞中过表达。最初，尝试使用常规方法如离子交换或凝胶过滤层析来纯化bcl-2蛋白。在这些纯化尝试中，我们确定bcl-2蛋白是高度疏水性的，并且易于聚集，这可能是预期的完整膜蛋白。通过离子交换层析和凝胶过滤层析，可以部分纯化该蛋白。为了将bcl-2纯化至表观均一性并避免聚集问题，我们使用针对选自人bcl-2的氨基酸序列的残基61-76的合成肽开发的单克隆抗体制备免疫亲和柱。将抗体偶联到CNBr活化的Sepharose 4 B或通过二甲基亚胺酸二盐酸盐交联成蛋白A-Sepharose。将相当于108 bcl-2过表达的昆虫细胞或淋巴细胞的细胞提取物应用于免疫亲和柱。经银染和免疫印迹分析，可回收约500 μg纯化的bcl-2蛋白。此外，将纯化的bcl-2蛋白电穿孔到前B淋巴细胞中，所述前B淋巴细胞不表达足够量的该蛋白以延迟糖皮质激素诱导的细胞凋亡的发生。电穿孔后的均匀纯的bcl-2蛋白，细胞被发现延长细胞的生存响应糖皮质激素。
The oncogene product bcl-2 functions as a repressor of programmed cell death and is a 26-kDa protein with a single predicted transmembrane segment located at the carboxyl terminus. The bcl-2 protein seems to function in different subcellular compartments, as evidenced by several biochemical and ultrastructural studies. The present study was performed to purify bcl-2 protein in significant quantities necessary for structural and functional studies. For this purpose, the bcl-2 gene was overexpressed in either baculovirus system or lymphocytes. Initially, attempts were undertaken to purify bcl-2 protein using conventional methods such as ion exchange or gel filtration chromatography. During these purification attempts we determined that bcl-2 protein is highly hydrophobic and prone to aggregation as might be expected for an integral membrane protein. By ion exchange and gel filtration chromatography, this protein could be partially purified. In order to purify bcl-2 to apparent homogeneity and avoid the aggregation problem, we prepared immunoaffinity columns using a monoclonal antibody developed against a synthetic peptide chosen from residues 61-76 of the amino acid sequence of human bcl-2. The antibody was either coupled to CNBr-activated Sepharose 4B or cross-linked into protein A-Sepharose by dimethylpimelimidate dihydrochloride. Cellular extract equivalent to 108bcl-2-overexpressing insect cells or lymphocytes was applied to immunoaffinity columns. Approximately 500 μg purified bcl-2 protein could be recovered as estimated by silver staining and immunoblotting. Furthermore, purified bcl-2 protein was electroporated into Pre-B lymphocytes which do not express this protein in sufficient quantity to delay the onset of glucocorticoid-induced apoptosis. Following electroporation of homogeneously pure bcl-2 protein, the cells were found to prolong cell survival in response to glucocorticoids.