Purification, crystallization and X-ray analysis of Pf-SCP (sarcoplasmic Ca-binding protein), related to storage and transport of calcium in mantle of Pinctada fucata.

Autor: Zhu L; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan., Wang L; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan., Matsuura A; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan., Zhang M; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan., Lu P; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan., Iimura K; Food Department, Department of Home Economics, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681, Japan., Nagata K; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan., Suzuki M; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. Electronic address: amichio@g.ecc.u-tokyo.ac.jp.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2021 Feb; Vol. 178, pp. 105781. Date of Electronic Publication: 2020 Oct 31.
DOI: 10.1016/j.pep.2020.105781
Abstrakt: Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and β = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules.
(Copyright © 2020 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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