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Crystals 2018, 8(7), 284; https://doi.org/10.3390/cryst8070284

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
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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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Abstract

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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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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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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