재조합 홍합 접착 단백질을 이용한 계면 접착 메커니즘 연구

Abstract

Marine adhesions gave many lessons to learn, and especially investigation on the underwater adhesives from marine-fouling organisms has proceeded as a development of potential underwater adhesives. The mussel adhesion is one of the best model systems. Marine mussels secret mussel adhesive proteins (MAPs) for their adhesion with fascinating properties such as strong adhesion to various material substrates, water displacement, biocompatibility, and controlled biodegradability. Investigation of mussel adhesion mechanism is regarded to be very important to understand mussel’s underwater adhesion and to develop various mussel-inspired materials for a wide range of applications as underwater bioadhesives. Difficult natural extraction and limited availability of MAPs have restricted various analyses. Particularly interfacial fp-3F and fp-5 MAPs, surface adhesion components found adjacent to the adhesion interface, are difficult to be naturally extracted and have extremely high Dopa contents. Recombinant protein expression was expected to contribute not only to replace natural extraction as an alternative strategy with sufficient productivity applicable for developing underwater bioadhesives but also to investigate mussel adhesion mechanism. In this thesis, we suggested mechanistic investigation of mussel adhesion with interfacial MAPs with the aid of advantages from recombinant MAPs: (1) production of recombinant protein is much easier than natural extraction of MAPs from mussel foots, (2) post-translational modification of recombinant MAPs can be adjusted, (3) recombinant expression provides homogeneous protein with only one type, and (4) recombinant MAPs are based on the natural sequences of MAPs.

First, we successfully produced Dopa-incorporated recombinant interfacial MAPs containing a large amount of Dopa using in vivo residue-specific unnatural amino acid incorporation method. The Dopa incorporation yield was over 90 % which is similar with natural interfacial MAPs (~100%), resulting high Dopa content of recombinant MAPs (~16.5 mol% for drfp-3F & ~23 mol% for drfp-5) as much as natural MAPs (~20 mol% for fp-3F & ~25 mol% for fp-5). It was confirmed that the Dopa-incorporated recombinant MAPs showed greatly enhanced surface adhesion in dry and underwater environments and strong water resistance.

Next, we observed Dopa-Fe3+ complexation of recombinant interfacial MAPs and its effects on both surface adhesive interaction and cohesive interaction based on the compatibility of Dopa-incorporated interfacial MAPs. Observation of Dopa-Fe3+ complexations of drfp-3F and drfp-5 suggested the possibility of Dopa-Fe3+ complexations at the plaque-substrate interface by natural fp-3F and fp-5. The force measurements using SFA analysis showed that intrinsic strong surface adhesion at low pH is dramatically reduced in the conditions where bis- and tris-Dopa-Fe3+ complexation occurred in higher pH and alternatively, strong cohesion is generated. It corresponds with the microenvironment changes during the mussel byssus formation. Thus, we proposed a possible mussel adhesion mechanism at the plaque-substrate interface, suggesting the potential role of Dopa-Fe3+ complexation as a regulator of Dopa functionality switching from surface adhesion to cohesion, in response to the microenvironment.

In addition, we examined and confirmed successful simple coacervate formation of interfacial MAPs with great assistance from mass production of recombinant interfacial MAPs, which showed the possibility of MAP simple coacervation. Using rfp-3F, we showed the salt-induced simple coacervation is possible at low pH corresponding to acidified environment of distal depression during mussel secretion. Rheological analysis confirmed viscous liquid properties and shear-thinning properties. SFA analysis confirmed stronger adhesion force compared to surface-deposited protein film. Thus, we could suggest that how properties of simple coacervate can contribute to mussel adhesion. In addition, possible driving forces were suggested based on the investigation of effects from Dopa, His6 tag, negatively charged amino acids, temperature, and salt types.

Collectively, we investigated the potential mussel adhesion mechanism using recombinant interfacial MAPs. Advantages from recombinant MAPs broaden the scope of research for mussel adhesion mechanism by overcoming the difficulties from limited availability of naturally extracted MAPs. In vivo residue-specific Dopa incorporation could produce Dopa-incorporated recombinant MAPs in E. coli which are very similar with natural MAPs, providing a good tool and materials for mechanistic research on mussel adhesion. Availability of both Dopa-deficient and Dopa-incorporated interfacial MAPs revealed the origins of Fe3+ complexation and could suggest a potential role of Dopa-Fe3+ complexation at interfacial region. High productivity of recombinant proteins enabled examination for simple coacervation of interfacial MAPs and finally, possibility of mussel simple coacervation could be suggested because simple coacervation of Dopa-incorporated recombinant MAPs are confirmed. In conclusion, mechanistic research on mussel adhesion with recombinant interfacial MAPs provided potential mussel adhesion mechanism at the plaque-substrate interface and showed its usability and ability to contribute to deepen understanding of mussel adhesion.