Production, extraction, and purification of human poly(ADP-ribose) polymerase-1 (PARP-1) with high specific activity.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in a range of activities associated with DNA metabolism and plays a key role in maintaining the integrity of DNA and chromatin structure. As such, this enzyme is likely to provide a useful target when using a rational drug design approach to develop pharmaceutical reagents, including cancer therapeutics. However, there is still a great deal to learn about the mode of action of PARP-1 and therefore efforts are being directed at gaining a better understanding of the relationship between its structure and function. To this end we have developed a rapid and relatively simple approach to producing and purifying PARP-1. Unlike traditional PARP-1 purification protocols, the method described here requires only one chromatography step thus minimizing losses of the enzyme and also avoids the use of a competitive inhibitor-based affinity chromatography step, which is common to several other protocols in the literature. The product of the method described here is high-quality native PARP-1 with a high specific activity and K(m) and V(max) values similar to what is reported by other workers in the field. This protocol is particularly well suited to making PARP-1 in a quantity and of a quality suitable for structure-function studies.
Knight
M I
MI
Centre for Bioprocessing and Food Technology, Victoria University of Technology, Melbourne, Australia.
Chambers
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011479:Protamines / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D018411:Spodoptera / Q000235:genetics; D014162:Transfection
D011479:Protamines; D011994:Recombinant Proteins; D006639:Histidine; D011065:Poly(ADP-ribose) Polymerases