Expression, Purification, and Functional Activity of Shell Matrix Protein Pearlin from the Pearl OysterPinctada fucata

Abstract

Nacre, also known as mother of pearl, contains factors that can promote osteogenesis. In this study, pearlin, a protein from the mantle tissue of the pearl oyster Pinctada fucata, was expressed and purified, and its functional activity analyzed using two mineralogenic cell lines, MRC-5 (fibroblasts) and MC3T3-E1 (preosteoblasts). The open reading frame of pearlin was subcloned into the expression vector pET32a( ) and used to transform Escherichia coli BL21 (DE3) strain. The expression of the recombinant protein (molecular weight: 34.19 kDa) was induced by isopropyl-β-D-thiogalactopyranoside in the form of inclusion bodies which were solubilized in 8 M urea. The recombinant protein was renatured by stepwise dialysis, purified by Ni-NTA affinity chromatography, and analyzed for the effects on cell proliferation by the MTT assay and osteoblastic differentiation by alkaline phosphatase (ALP) activity. The recombinant pearlin promoted proliferation ofMRC-5 andMC3T3-E1 cells at 10 µg/mL and increased their ALP activity at 2.5 µg/mL and 5 µg/mL, respectively. The present study showed that the recombinant pearlin exerted functional effects similar to those of the natural protein, laying a foundation for large-scale production of pearlin in a prokaryotic system.