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A rapid purification procedure for SV40 large T antigen has been developed which combines the use of an adenovirus-SV40 hybrid virus which overproduces large T antigen, and immunoaffinity chromatography on an anti-large T monoclonal antibody coupled to protein A Sepharose. The protein exhibits the p53-binding, ATPase, and sequence-specific DNA-binding activities of T antigen. The purification procedure can be completed in 1 day and allows the isolation of milligram amounts of large T in excellent yield. The pure protein is extremely antigenic and is tolerant of iodination to high specific activity, permitting the development of a competition radioimmunoassay for large T that reliably detects nanogram amounts of the protein.