کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2021062 | 1069225 | 2011 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase](/preview/png/2021062.png)
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 2 mg per 1 L 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.
Journal: Protein Expression and Purification - Volume 75, Issue 2, February 2011, Pages 230–235