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.

Pf-SCP是一种具有两个EF手基序和磷酸化位点的21 kDa蛋白，已从地幔组织中鉴定出并与钙离子结合并将钙成分从细胞转运到Pinctada fucata的壳中。为了揭示Pf-SCP钙结合活性的分子基础，我们在大肠杆菌中表达了全长Pf-SCP的重组蛋白。通过Ni亲和色谱和尺寸排阻色谱纯化的重组Pf-SCP（rPf-SCP）在SDS-PAGE上显示为单条带。圆二色光谱显示，当rPf-SCP与钙离子和碳酸钙相互作用时，α-螺旋含量降低。Western印迹和免疫染色验证了Pf-SCP在壳中的表达以及在上皮上皮细胞中的大多数定位。为了进一步了解钙离子和碳酸钙对Pf-SCP的结构和功能的调控，进行了在钙离子存在下rPf-SCP的结晶实验。在钙离子存在下获得的rPf-SCP晶体衍射X射线，分辨率高达1.8。该晶体的空间群为C2与晶胞参数一 = 96.828埃，b  = 55.906埃，c ^  = 102.14埃，β  = 90.009°，表明RPF-SCP的三个分子中含有一个不对称单位作为估计马修斯系数的值。这些结果表明，Pf-SCP可能通过浓缩地幔上皮细胞内部的钙离子并与碳酸钙分子相互作用而在钙离子运输和壳矿化中发挥作用。