Purification of recombinant p53 from Sf9 insect cells.
We describe a method for purifying recombinant p53 from baculovirus infected cells in one step by anion exchange chromatography. The p53 is full-length with no flanking sequences and its expression is driven by the baculovirus polyhedron promoter. We also describe how to concentrate the p53 up to 0.9 mg/mL. By gel filtration analysis, we demonstrate that 20% of the p53 forms a tetramer, and 80% forms a monomer. In a DNA binding assay known as the electromobility shift assay, the purified p53/DNA complex forms a single band the gel. This simple procedure should be useful for investigations into the biochemistry of the p53 protein.
Sun
Xiu Zhu
XZ
Department of Chemistry and Biochemistry, California State University at Los Angeles, USA.
Nguyen
John
J
Momand
Jamil
J
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007313:Insecta; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA