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홍합접착단백질을 이용한 복합 코아세르베이트에 관한 연구

홍합접착단백질을 이용한 복합 코아세르베이트에 관한 연구
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Mussels inhabit on rocky seashore using foot proteins (fps), which strongly adhere on various surfaces within wet environment. It is well known and studied that mussel adhesive proteins (MAPs) adhere in wet surfaces and harden mostly using L-3,4-dihydroxyphenyl alanine (dopa). Becuase MAPs exist condensed form in mussel foot, it has been suggested that complex coacervation (liquid-liquid phase separation via concentration) might be involved in the highly condensed and non-water dispersed adhesion process of MAPs. However, as suffecient amount of purified natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation model. In the present work, we demonstrated complex coacervation in a system including recombinant MAPs and several anionic partners. The recombinant hybrid MAPs, fp-151 and fp-131, and natural MAP types, partial foot protein type 1, type 3, and type 5, were mass-produced in our research team. Thus, we observed successful complex coacervation using all recombinant MAPs and the anionic partners including hyaluronic acid (HA). We found that foot protein type 4 (fp-4) is composed of basic and acidic part (fp-4a). Thus, we decied to use fp-4a as acidic partner for complex coacervation with recombinant MAPs. Succesful formation of complex coacervation between MAPs was confirmed by turbidity measurement and morphology observation. Moreover, we observed that coacervated MAPs have porous structure similar to the mediated region of mussel plaque, where MAPs placed. Coacervation is known for having extremely low interfacial tension. Thus, complex coacervation system has been utilized on microencapsulation. The aggregation of hybrid MAP, fp-151 was studied by Hofmeister salt series, which are continuously divided into chaotropes and kosmotropes by hydration force. Generally, kosmotropes show salting-out phenomenon by their high charge density and chaotropes show salting-in behavior. As a result, aggregation degree of fp-151 increased by chaotropic salts. Because it was known that aggregation of polyelectrolyte and its interfacial tension are directly connected, aggregation tendency and interfacial tension of fp-151/HA coacervate was characterized on Hofmeister salt series. Aggregation degree of fp-151/HA also increased by chaotropic salts. We found that its interfacial tension increased by chaotropic salts. The features may apply on finding suitable condition for various applications such as coacervation with other anionic biomacromolecules or microencapsulation. The components of microencapsulation of oil droplets using a MAP-based coacervation, fp-151/HA and fp-151/heparin, were successfully confirmed by optical and fluorescence microscopes, but chondroitin system did not form. Microcapsule with hydrophobic oil maintained for 8 - 21 days. Besides, hydrophilic biomolecules, such as GFP, basic fibroblast growth factor (bFGF), and vancomycin, were loaded into fp-151/HA coacervate. As a model system, GFP loaded fp-151/HA coacervates was confirmed to have adhesive and no-dispersion propertes in water. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strengtsh of MAPs in both dry and underwater environments. Collectively, complex coacervations of recombinant MAPs were confirmed, and the condensation and porous structure formatiom of the MAP coacervates gave a clue for mussel adhesion mechanism. Moreover, the results demonstrate that a complex coacervation system based on MAPs shows superior adhesive properties, combined with additional valuable features, including liquid/liquid phase separation, microencapsulation, and complexation. Therefore, our proposed novel drug delivery system candidate based on MAP complex coacervation could be useful in the development of new adhesive biomaterials, including multifunctional drug carriers, for use in biotechnological and biomedical applications.
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