Control of nacre biomineralization by Pif80 in pearl oyster.

Molluscan nacre is a fascinating biomineral consisting of a highly organized calcium carbonate composite that provides unique fracture toughness and an iridescent color. Organisms elaborately control biomineralization using organic macromolecules. We propose the involvement of the matrix protein Pif80 from the pearl oyster Pinctada fucata in the development of the inorganic phase during nacre biomineralization, based on experiments using the recombinant form of Pif80. Through interactions with calcium ions, Pif80 participates in the formation of polymer-induced liquid precursor-like amorphous calcium carbonate granules and stabilizes these granules by forming calcium ion-induced coacervates. At the calcification site, the disruption of Pif80 coacervates destabilizes the amorphous mineral precursors, resulting in the growth of a crystalline structure. The redissolved Pif80 controls the growth of aragonite on the polysaccharide substrate, which contributes to the formation of polygonal tablet structure of nacre. Our findings provide insight into the use of organic macromolecules by living organisms in biomineralization.

Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals.

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 Ca(2+). Ca(2+)-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.

Highly purified mussel adhesive protein to secure biosafety for in vivo applications.

BACKGROUND: Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult. RESULTS: In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151. CONCLUSIONS: Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.