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Recombinant p53 displays heterogeneity during isoelectric focusing
Author(s) -
Heukeshoven Jochen,
März Annette,
Warnecke Gabriele,
Deppert Wolfgang,
Tolstonog Genrich V.
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200205
Subject(s) - isoelectric focusing , recombinant dna , chemistry , chromatography , biochemistry , gene , enzyme
Human recombinant, baculovirus‐expressed p53 protein focuses on 2 D gels in multiple spots in the narrow p I range. Re‐electrophoresis of the individual spots resulted in the appearance of multiple spots. The strings of spots were neither species specific, nor characteristic for baculovirus‐expressed p53. Moreover, mutant p53 did not deviate from wild‐type p53, indicating that this is an inherent property of p53. Okadaic acid treatment of insect cells, phosphate substitution reaction of purified p53, and individual analysis of all spots by mass spectrometry revealed that only a fraction of the recombinant p53 is phosphorylated. This finding excluded that the individual p53 spots in 2 D gels reflect charge isomers generated by phosphorylation, but rather suggest that they are due to conformational flexibility of urea‐denatured monomeric p53 molecules or deamidation of asparagine and glutamine residues. The latter possibility was confirmed by N ano LC ‐ ESI MS / MS analysis. Our data provide a putative hint for a novel regulatory level for function and stability of p53, particularly the long‐lived mutant p53 overexpressed in diverse tumor types.