So Yeong Bahn

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

BackgroundUnique 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.ResultsIn 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.ConclusionsThrough 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.

Control of nacre biomineralization by Pif80 in pearl oyster

Pif80 is a key protein for the formation and organization of mineral precursor in pearl oyster nacre biomineralization. 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.

CaCO3 thin-film formation mediated by a synthetic protein-lysozyme coacervate

Coacervation is a liquid–liquid phase separation process of macromolecular polyelectrolytes. The formation of simple and complex coacervates of a synthetic acidic protein, GG1234, as a model shell matrix protein, was investigated using turbidity measurements and microscopic morphological observations. Simple coacervation of GG1234 was optimally induced at pH 3.75 and below 50 mM for all of the tested salts, and complex coacervates were prepared at pH 4–9 in sodium acetate solution at various ratios of GG1234 to lysozyme without inducing simple coacervation. The complex coacervates also had the ability to microencapsulate hydrophobic oil droplets in a similar manner to that of other complex coacervation systems. Remarkably, a thin film was formed through in vitro CaCO3 crystallization in the presence of complex coacervates, which was expected to be planar and poorly crystalline CaCO3 guided at the interface of two immiscible liquid phases upon complex coacervation. Collectively, our results indicate that our synthetic acidic matrix protein can be used as the main protein for simple and complex coacervations, and the coacervates of this protein may be involved in thin film formation in CaCO3 crystallization.