Over-expression in E. coli and purification of functional full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein)

Jaramillo-Tatis, Sergio; Bamm, Vladimir V.; Vassall, Kenrick A.; Harauz, George

doi:10.1016/j.pep.2014.05.013

Cited by 1 publication

(1 citation statement)

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“…The preparation of the enzyme source for the SCP1 in the mammalian system is not reported. The production of a full-length version of recombinant SCP1 in the functional form under native conditions was not possible, primarily because of solubility issues (24) . Furthermore, even purifying the full-length form of the hSCP1 from E. coli expression system has also not been possible previously for another reason, because the protein consistently underwent proteolysis along the N-terminus during the expression (16) .…”

Section: Resultsmentioning

confidence: 99%

In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure

Koo¹,

Bahk²

2014

BMB Reports

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RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of β-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain insight into the PTM of hSCP1, we used the Western blot, immunoprecipitation, succinylayed wheat germ agglutininprecipitation, liquid chromatography-mass spectrometry analyses, and site-directed mutagenesis and identified the Ser41 residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s). [BMB Reports 2014; 47(10): 593-598]

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Section: Resultsmentioning

confidence: 99%