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.
Chalkley
G E
GE
Protein Isolation and Cloning Laboratory, Imperial Cancer Research Fund, London, England.
Knowles
P P
PP
Whitehead
P C
PC
Coffer
A I
AI
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics*; D002413:Cations, Divalent; D002478:Cells, Cultured; D002482:Cellulose / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003035:Cobalt / Q000378:metabolism; D004247:DNA; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D046911:Macromolecular Substances; D009036:Moths; D009532:Nickel / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; C033190:DNA-cellulose; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D003035:Cobalt; D009532:Nickel; D002482:Cellulose; D004247:DNA; D015032:Zinc