Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system.
We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies.
Giner
H
H
Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie, Heidelberg, Germany.
Simonin
F
F
de Murcia
G
G
Ménissier-de Murcia
J
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D005784:Gene Amplification; D006801:Humans; D009036:Moths; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D015032:Zinc