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/l culture. Milligram amounts of p53 were purified to apparent homogeneity using chromatography on double‐stranded DNA‐cellulose (∼58% yield) followed by immunoaffinity chromatography with an epitope elution step (up to 48% yields) at 4°C. The M r of extracted p53 both from insect cell lysates and after purification was 54000 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 Zn 2+ , Ni 2+ and Co 2+ with high affinity.