[In vitro binding of p53 and telomeric repeat factor 2].
OBJECTIVE
To clarify the regulation of p53 through telomere pathway by investigating the molecular interaction between p53 and the main telomere-associated protein Telomeric Repeat Factor 2 (TRF2) in vitro.


METHODS
Four different p53-GST (glutathione S-transferase) fusion proteins and GST were expressed in E. coli and purified through glutathione sepharose 4B beads. The human recombinant p53s included wild type p53 (1-393), N terminus-truncated form p53 2C (95-393), C terminus-truncated form p53 N5 (2-293) and single amino acid mutant p53 R175H (175 arginine to histidine). Purified p53-GST fusion proteins and GST were mixed with cellular protein extracts of human breast cancer cells MCF-7 in vitro by pull down. The molecular interaction between p53 and TRF2 were detected by Western blot.


RESULTS
SDS-PAGE and Coomassie brilliant blue staining showed that the molecular weights of all purified proteins were as expected, with purities over 90%. Western blot of TRF2 indicated that both wild type p53 and p53 R175H could bind with TRF2 of MCF-7 cells in similar capacity, while GST alone failed to do so. The molecular interaction between p53 2C and TRF2 was enhanced. In contrast, the interaction between p53 N5 and TRF2 was significantly reduced.


CONCLUSIONS
p53 can interact with TRF2 directly and specifically in vitro, with C terminus of p53 (293-393) being the binding region for their interaction. This C terminus-dependent interaction between p53 and TRF2 may be related to the cellular activities induced by telomere alterations.
Li
Ling
L
Department of Pathology, Health Science Center, Peking University, Beijing 100083, China. lingli@bjmu.edu.cn
Zhang
Bo
B
Zou
Wan-Zhong
WZ
Zheng
Jie
J
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D035341:Telomeric Repeat Binding Protein 2 / Q000378:metabolism*; D014170:Transformation, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D035341:Telomeric Repeat Binding Protein 2; D016159:Tumor Suppressor Protein p53; D005982:Glutathione Transferase