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Crystals 2018, 8(7), 284;

Over-Expression, Secondary Structure Characterization, and Preliminary X-ray Crystallographic Analysis of Xenopus tropicalis Ependymin

School of Systems Biomedical Science, Soongsil University, Seoul 06978, Korea
Author to whom correspondence should be addressed.
Received: 19 June 2018 / Revised: 6 July 2018 / Accepted: 9 July 2018 / Published: 11 July 2018
(This article belongs to the Special Issue Biological Crystallization)
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The gene encoding frog (Xenopus tropicalis) ependymin without the signaling sequence was gene-synthesized, and the protein successfully over-expressed in ~mg quantities adequate for crystallization using insect cell expression. Circular dichroism (CD) analysis of the protein purified with >95% homogeneity indicated that ependymin contains both α-helix and β-strand among the secondary structure elements. The protein was further crystallized using polyethylene glycol 8000 as the precipitating reagent, and X-ray diffraction data were collected to 2.7 Å resolution under cryo-condition at a synchrotron facility. The crystal belongs to a hexagonal space group P6122 (or P6522) having unit cell parameters of a = b = 61.05 Å, c = 234.33 Å. Matthews coefficient analysis indicated a crystal volume per protein mass (VM) of 2.76 Å3 Da−1 and 55.4% solvent content in the crystal when the calculated molecular mass of the protein only was used. However, the apparent SDS-PAGE molecular mass of ~33 kDa (likely resulting from N-glycosylation) suggested VM of 1.90 Å3 Da−1 and 35.4% solvent content instead. In both cases, the asymmetric unit of the crystal likely contains only one subunit of the protein. View Full-Text
Keywords: ependymin (EPN); ependymin-related protein (EPDR); mammalian ependymin-related protein (MERP) ependymin (EPN); ependymin-related protein (EPDR); mammalian ependymin-related protein (MERP)

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Park, J.K.; Sim, Y.W.; Park, S. Over-Expression, Secondary Structure Characterization, and Preliminary X-ray Crystallographic Analysis of Xenopus tropicalis Ependymin. Crystals 2018, 8, 284.

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