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

Abstract

The gene encoding frog (Xenopus tropicalis) ependymin without the signaling sequence was gene-synthesized, and the protein successfully over-expressed in similar to 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 alpha-helix and beta-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 angstrom resolution under cryo-condition at a synchrotron facility. The crystal belongs to a hexagonal space group P6(1)22 (or P6(5)22) having unit cell parameters of a = b = 61.05 angstrom, c = 234.33 angstrom. Matthews coefficient analysis indicated a crystal volume per protein mass (V-M) of 2.76 angstrom(3) 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 similar to 33 kDa (likely resulting from N-glycosylation) suggested V-M of 1.90 angstrom(3) 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.