A NONDENATURING PURIFICATION SCHEME FOR THE DNA-BINDING DOMAIN OF POLY(ADP-RIBOSE) POLYMERASE, A STRUCTURE-SPECIFIC DNA-BINDING PROTEIN

Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA re pair given its ability to recognize and bind to DNA strand breaks. Dur ing apoptosis, PARP is proteolytically cleaved into two stable fragmen ts, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa frag ment containing the automodification and catalytic domains, To underst and the DNA-binding properties of PARP, we expressed a recombinant hex ahistidine tagged protein (His-DBD) in Escherichia coli. We modified e xpression to facilitate protein folding by including zinc and reducing the induction temperature, Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner. To eliminate contamination with bacterial DNA that occurred d uring the purification process, a purification procedure was developed to produce DNA-free protein. In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out w hile the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleava ge with no appreciable loss to unwanted proteolytic degradation. This nondenaturing purification scheme results in high-quality, native PARP -DBD for use in structural and biochemical studies. (C) 1998 Academic Press.