Expression and purification of active recombinant ATM protein from transiently transfected mammalian cells.
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.
Rhodes
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N
Oncology Biology Department, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, USA.
Gilmer
T M
TM
Lansing
T J
TJ
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins; D006801:Humans; D009842:Oligopeptides; D010455:Peptides / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C085706:FLAG peptide; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases