Biochemical characterisation of purified human wild-type p53 overexpressed in insect cells.

Conditions for the overexpression of human wild-type p53 using a baculovirus construct were optimised in insect cells which produced up to 20 mg p53/1 culture. Milligram amounts of p53 were purified to apparent homogeneity using chromatography on double-stranded DNA-cellulose (approximately 58% yield) followed by immunoaffinity chromatography with an epitope elution step (up to 48% yields) at 4 degrees C. The M(r) of extracted p53 both from insect cell lysates and after purification was 54,000 by SDS/PAGE. Isoelectric focusing showed recombinant p53 to be an acidic protein, focusing at pI 6.0 under non-denaturing conditions. Expressed p53 at all stages of purification reacted by immunoblotting with specific p53 monoclonal antibodies, indicating the presence of intact epitopes at the C-terminus, N-terminus and central region of the protein. From ultracentrifugation studies, pure p53 exhibited significant oligomerisation, and sedimented broadly within the 7-12-S region of sucrose gradients. Pure p53 slowly precipitated out of solution at concentrations between 1-6 mg/ml even in the presence of 1% detergent. Using metal affinity chromatography, we have established that pure p53 binds the immobilised divalent ions Zn2+, Ni2+ and Co2+ with high affinity.