Recombinant p53 displays heterogeneity during isoelectric focusing.

Details

Serval ID
serval:BIB_3DBC791FCD19
Type
Article: article from journal or magazin.
Collection
Publications
Title
Recombinant p53 displays heterogeneity during isoelectric focusing.
Journal
Electrophoresis
Author(s)
Heukeshoven J., März A., Warnecke G., Deppert W., Tolstonog G.V.
ISSN
1522-2683 (Electronic)
ISSN-L
0173-0835
Publication state
Published
Issued date
09/2012
Peer-reviewed
Oui
Volume
33
Number
18
Pages
2818-2827
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
Human recombinant, baculovirus-expressed p53 protein focuses on 2D gels in multiple spots in the narrow pI 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 2D 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 NanoLC-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.
Keywords
Amino Acid Sequence, Animals, Baculoviridae/genetics, Cell Line, Electrophoresis, Gel, Two-Dimensional, Humans, Isoelectric Focusing/methods, Mice, Molecular Sequence Data, Moths, Phosphorylation, Recombinant Proteins/chemistry, Recombinant Proteins/genetics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tumor Suppressor Protein p53/chemistry, Tumor Suppressor Protein p53/genetics
Pubmed
Web of science
Create date
15/12/2017 16:58
Last modification date
09/01/2020 7:26
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