Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase.
Poly(ADP-ribosyl)ation, which is mainly involved in DNA repair and replication, is catalyzed mainly by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Although recombinant human PARP-1 (hPARP-1) is commercially available, there are no reports on the preparation of recombinant human PARG (hPARG). Here, we report the efficient expression and purification of a recombinant hPARG-catalytic domain (hPARG-CD) from Escherichia coli (E. coli). hPARG-CD was expressed as a fusion protein with a glutathione S-transferase (GST) tag at the N-terminus and a hexahistidine (6His) tag at the C-terminus. Both high cell density and low temperature culture conditions were important for the maximum production of soluble recombinant hPARG-CD. After sequential affinity chromatography using immobilized metal affinity resin and glutathione-Sepharose (GSH-Sephasrose), more than 95% pure recombinant hPARG-CD was obtained with a yield of approximately 2mg per 1L of E. coli culture medium. The km and Vmax values of purified recombinant hPARG-CD were 9.0 μM and 35.6 μmol/min/mg protein, respectively. These kinetic values were similar to those of purified endogenous hPARG reported previously. Furthermore, the recombinant hPARG-CD was inhibited by known PARG inhibitors such as adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), eosin Y, and phloxine B. These results show that the recombinant hPARG-CD is useful to search for specific inhibitors and to elucidate the regulatory mechanisms of hPARG.
Okita
Naoyuki
N
Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan. nokita7@rs.noda.tus.ac.jp
Ohta
Ryo
R
Ashizawa
Daisuke
D
Yamada
Yohichiro
Y
Abe
Hideaki
H
Abe
Takehiko
T
Tanuma
Sei-Ichi
S
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004260:DNA Repair; D004261:DNA Replication; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*
D004791:Enzyme Inhibitors; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D005982:Glutathione Transferase; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase