Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase

Protein Expr Purif. 2011 Feb;75(2):230-5. doi: 10.1016/j.pep.2010.09.012. Epub 2010 Sep 25.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA Repair
  • DNA Replication
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli
  • Glutathione Transferase / chemistry
  • Glutathione Transferase / metabolism
  • Glycoside Hydrolases* / antagonists & inhibitors
  • Glycoside Hydrolases* / biosynthesis
  • Glycoside Hydrolases* / genetics
  • Glycoside Hydrolases* / isolation & purification
  • Histidine / chemistry
  • Histidine / metabolism
  • Humans
  • Kinetics
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / antagonists & inhibitors
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / isolation & purification*

Substances

  • Enzyme Inhibitors
  • His-His-His-His-His-His
  • Oligopeptides
  • Recombinant Fusion Proteins
  • Histidine
  • Glutathione Transferase
  • Glycoside Hydrolases
  • poly ADP-ribose glycohydrolase