Recombinant p53 displays heterogeneity during isoelectric focusing
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Recombinant p53 displays heterogeneity during isoelectric focusing. / Heukeshoven, Jochen; März, Annette; Warnecke, Gabriele; Deppert, Wolfgang; Tolstonog, Genrich V.
in: ELECTROPHORESIS, Jahrgang 33, Nr. 18, 18, 09.2012, S. 2818-2827.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Recombinant p53 displays heterogeneity during isoelectric focusing
AU - Heukeshoven, Jochen
AU - März, Annette
AU - Warnecke, Gabriele
AU - Deppert, Wolfgang
AU - Tolstonog, Genrich V
N1 - © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2012/9
Y1 - 2012/9
N2 - 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.
AB - 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.
KW - Animals
KW - Humans
KW - Mice
KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Phosphorylation
KW - Cell Line
KW - Electrophoresis, Gel, Two-Dimensional
KW - Isoelectric Focusing/methods
KW - Baculoviridae/genetics
KW - Moths
KW - Recombinant Proteins/chemistry/genetics
KW - Tumor Suppressor Protein p53/chemistry/genetics
KW - Animals
KW - Humans
KW - Mice
KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Phosphorylation
KW - Cell Line
KW - Electrophoresis, Gel, Two-Dimensional
KW - Isoelectric Focusing/methods
KW - Baculoviridae/genetics
KW - Moths
KW - Recombinant Proteins/chemistry/genetics
KW - Tumor Suppressor Protein p53/chemistry/genetics
U2 - 10.1002/elps.201200205
DO - 10.1002/elps.201200205
M3 - SCORING: Journal article
C2 - 23019099
VL - 33
SP - 2818
EP - 2827
JO - ELECTROPHORESIS
JF - ELECTROPHORESIS
SN - 0173-0835
IS - 18
M1 - 18
ER -
