Determination of unknown poly(ADP-ribosyl)ated proteins and binding sites

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We identified tow proteins that are poly(ADP-ribosyl)ated in vivo. They are p53 and nucleophosmin/B23. To identify the site of poly(ADP-ribosyl)ation of p53, we made the expression vector to produce GST-p53 fusion protein in E. coli. We confirmed the poly(ADP-ribosyl)ation of the entire product of p53 (aa1-aa393) by PARP-1. Currently we are preparing several GST-truncated p53 proteins, that lack about 40 amino acids, and analyzing poly(ADP-ribosyl)ated domain of p53.We identified a GST-truncated p53 protein, in which about 40 amino acids are deleted in the DNA binding domain, that lacks the acceptor activity of poly(ADP-ribosyl)ation. To identify the poly(ADP-ribosyl)ated proteins in general, we made an affinity column that have the antibody against poly(ADP-ribose), 1OH. We are optimizing the condition to bind the proteins from centrosome fractions and from other cellular fractions. To prepare a large amount of proteins that are poly(ADP-ribosylated), we are collecting the proteins from Drosophila mutant that lacks poly(ADP-ribose) glycohydrolase.