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 predominan tly a nuclear protein where it interacts with p53 and c-Abl as part of a radiation signal transduction pathway(s). We describe here the clon ing of full-length ATM cDNA in a baculovirus vector to produce recombi nant 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 purif ication of the protein by affinity chromatography. The protein yield w as 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 immunoprecipita tes with antibodies directed against the hexahistidine tag sequence. T hese results demonstrate that ATM can be expressed inefficiently in ba culovirus infected insect cells and the data suggest that it phosphory lates itself. (C) 1998 Academic Press.