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 repair given its ability to recognize and bind to DNA strand breaks. During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains. To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli. We modified expression 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 during 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 while the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage 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.
Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico, 87545, USA.
T
T
Ruscetti
J
J
Newman
T S
TS
Peat
J
J
Francis
R
R
Nolan
T C
TC
Terwilliger
S R
SR
Peterson
B E
BE
Lehnert
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013917:Thrombin
D017931:DNA Primers; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases; D013917:Thrombin