Expression and purification of active recombinant ATM protein from transiently transfected mammalian cells.
Expression and purification of active recombinant ATM protein from transiently transfected mammalian cells.
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从瞬时转染的哺乳动物细胞中表达和纯化活性重组 ATM 蛋白。
DOI：
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发表时间：
2001
影响因子：
1.6
通讯作者：
T. J. Lansing
中科院分区：
文献类型：
--
作者：
N. Rhodes;T. Gilmer;T. J. Lansing
在人类疾病共济失调毛细血管扩张症（AT）中突变的基因，称为ATM，编码一种参与DNA修复和细胞周期控制的大蛋白激酶。ATM功能的生物化学表征有些困难，因为它的大尺寸（约370 kDa）和相对较低的表达水平在几个系统。大多数研究使用免疫沉淀ATM或通过相对复杂的程序获得的纯化ATM。在这里，我们描述了一种有效的方法表达和纯化FLAG表位标记的重组人ATM蛋白（F-ATM）。该方法利用F-ATM在瞬时转染的293 T细胞中的表达，随后进行抗FLAG-琼脂糖亲和层析。转染程序已被优化为大（225-cm（2））培养瓶和F-ATM可以纯化到接近同质的SDS-PAGE判断。该过程产生约1微克催化活性F-ATM蛋白/225-cm（2）烧瓶，可用于生物化学研究。
The gene mutated in the human disease ataxia telangiectasia (AT), termed ATM, encodes a large protein kinase involved in DNA repair and cell cycle control. Biochemical characterization of ATM function has been somewhat difficult because of its large size (approximately 370 kDa) and relatively low level of expression in several systems. The majority of studies have used immunoprecipitated ATM or purified ATM obtained through relatively complex procedures. Here, we describe an efficient method for the expression and purification of FLAG-epitope-tagged recombinant human ATM protein (F-ATM). This method utilizes the expression of F-ATM in transiently transfected 293T cells followed by anti-FLAG-agarose affinity chromatography. The transfection procedure has been optimized for large (225-cm(2)) culture flasks and F-ATM can be purified to near homogeneity as judged by SDS-PAGE. This procedure yields approximately 1 microg of catalytically active F-ATM protein/225-cm(2) flask that can be used for biochemical studies.
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DOI：
10.1073/pnas.96.20.11134
发表时间：
1999-09-28
影响因子：
11.1
作者：
Smith, GCM;Cary, RB;Jackson, SP
通讯作者：
Jackson, SP
影响因子：
56.9
作者：
Cortez, D;Wang, Y;Elledge, SJ
通讯作者：
Elledge, SJ
影响因子：
3.5
作者：
SAVITSKY, K;SFEZ, S;ROTMAN, G
通讯作者：
ROTMAN, G
DOI：
10.1073/pnas.190030497
发表时间：
2000-09-12
影响因子：
11.1
作者：
Matsuoka, S;Rotman, G;Elledge, SJ
通讯作者：
Elledge, SJ
影响因子：
56.9
作者：
SAVITSKY, K;BARSHIRA, A;SHILOH, Y
通讯作者：
SHILOH, Y