Cloning and expression of the ataxia-telangiectasia gene in baculovirus.
The gene mutated in the human genetic disorder ataxia-telangiectasia, ATM, is implicated in the response to radiation-induced DNA damage and to a more widespread signalling defect. The ATM protein is predominantly a nuclear protein where it interacts with p53 and c-Abl as part of a radiation signal transduction pathway(s). We describe here the cloning of full-length ATM cDNA in a baculovirus vector to produce recombinant protein. Expression of ATM, as a soluble protein, was observed by 36 h post-infection using immunoblotting with anti-ATM antibody. The presence of a hexahistidine tag on ATM was used as the basis for purification of the protein by affinity chromatography. The protein yield was only 20 ng/100 ml of infected cells, presumably because of the size of the protein and adverse effects on cell growth when overexpressed. ATM was found to have autophosphorylation activity in immunoprecipitates with antibodies directed against the hexahistidine tag sequence. These results demonstrate that ATM can be expressed inefficiently in baculovirus infected insect cells and the data suggest that it phosphorylates itself.
Scott
S P
SP
The Queensland Cancer Fund Research Laboratories, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, 4029, Australia.
Zhang
N
N
Khanna
K K
KK
Khromykh
A
A
Hobson
K
K
Watters
D
D
Lavin
M F
MF
D000818:Animals; D001260:Ataxia Telangiectasia / Q000235:genetics*; D064007:Ataxia Telangiectasia Mutated Proteins; D016367:Baculoviridae / Q000235:genetics*; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000235:genetics; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases