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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 30, Number 5
BKCSDE 30(5)
May 20, 2009 

 
Title
Purification and NMR Studies of RNA Polymerase II C-Terminal Domain Phosphatase 1 Containing Ubiquitin Like Domain
Author
Sunggeon Ko, Youngmin Lee, Jong Bok Yoon, Weontae Lee*
Keywords
RNA Polymerase II, CTD-Phosphatase, NMR, Cloning, Purification
Abstract
RNA polymerase II C-terminal domain phosphatase 1 containing ubiquitin like domain (UBLCP1) has been identified as a regulatory molecule of RNA polymerase II. UBLCP1 consists of ubiquitin like domain (UBL) and phosphatase domain homologous with UDP and CTD phosphatase. UBLCP1 was cloned into the E.coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using both affinity and gel-filtration chromatography. Domains of UBLCP1 protein were successfully purified as 7 mg/500 mL (UBLCP1, 36.78 KDa), 32 mg/500 mL (UBL, 9 KDa) and 8 mg/500 mL (phosphatase domain, 25 KDa) yielded in LB medium, respectively. Isotope-labeled samples including triple-labeled (2H/15N/13C) UBLCP1 were also prepared for hetero-nuclear NMR experiments. 15N-1H 2D-HSQC spectra of UBLCP1 suggest that both UBL and phosphatase domain are properly folded and structurally independent each other. These data will promise us further structural investigation of UBLCP1 by NMR spectroscopy and/or X-ray crystallography.
Page
1039 - 1042
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