Abstract
Objectives
To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO3 biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized.
Results
A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l−1 with a purity of >95 %. It efficiently formed a complex with Ca2+. Ca2+-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20–30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein.
Conclusions
Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.
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Acknowledgments
This work is supported by the National Research Foundation of Korea Grant, which is funded by the Ministry of Science, ICT and Future Planning, Korea (NRF-C1ABA001-2011-0029960 and NRF-2012R1A1A1003516; to Y.S.C.) and by the Marine Biotechnology Program (Marine BioMaterials Research Center), which is funded by the Ministry of Oceans and Fisheries, Korea (to H.J.C. & Y.S.C.).
Supporting information
Supplementary Fig. 1—Schematic representation of the amino acid sequence of BA (NCBI reference sequence; BAA20465). Black underline indicates a signal peptide. The yellow box is the shematrin-2-like domain and the green box is a potential calcium-binding domain. Red letters indicate acidic amino acid residues and blue letters indicate basic amino acid residues.
Supplementary Fig. 2—a Overall scheme of the primary structures of GRP_BA and recombinant GRP_BA protein (rBA); b the optimized sequence of GRP_BA without N-terminal signal sequence for over-expression.
Supplementary Fig. 3—Wide scan X-ray photoelectron spectrum (XPS) of CaCO3 precipitates obtained at 4 °C after a 20 h incubation using the ammonium carbonate vaporization method (V–L) with 1 mg rBA ml-1.
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Song, W., Bahn, S.Y., Cha, H.J. et al. Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals. Biotechnol Lett 38, 809–816 (2016). https://doi.org/10.1007/s10529-016-2039-x
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DOI: https://doi.org/10.1007/s10529-016-2039-x