Studies On The Interfacial Adhesion Mechanisms And Peptide Mimics Of Balanus Albicostatus Cp9k

In the long process of evolution,some marine creatures have developed the unique ability to tenaciously adhere to almost any surfaces in water.These organisms,although whose underwater attachment leads to the marine biofouling issue which has been bedeviling humans for millennia,provide ideal biological prototypes for the design of bio-inspired adhesives that can function underwater.Here,in the thesis we focus on the adhesive protein Balcp19 k secreted by Balanus albicostatus,a widely-spread marine fouling species along China's coasts,to study its physicochemical properties,adhesive functions,and biomimetic applications.Using the Escherichia coli expression system,we previously produced a fusion protein named “Trx-Balcp19k”,which bears an additional thioredoxin(Trx)tag to help the two conserved Cys of Balcp19 k form an intramolecular disulfide bond.Here,in order to obtain tag-free recombinant Balcp19k(rBalcp19k)for further studies,all extra tags in the fusion protein are removed by specific enzymatic digestion and the target protein is purified by integrating multiple chromatograph techniques.Compared to the native Balcp19 k,the prepared tag-free rBalcp19 k only introduces two additional amino acids at the N-termini.Interestingly,during preparing recombinant proteins,it is found that the fusion protein Trx-Balcp19 k can self-aggregate to form a super sticky gel-like material under appropriate conditions,without adding any other biochemical components.In air,this material displays surprisingly high adhesion strength on metals and even rivals some commercial glue.It also has good biocompatibility and shows potential applications in biomedical areas.To understand the interfacial adhesion mechanisms of cp19 k,subsequently,we conduct a series of in vitro studies to examine the structures,self-assembly properties,and adhesive functions of rBalcp19 k under different conditions.For the first time,we find that rBalcp19 k possesses time-dependent secondary structure transition,as well as the self-assembling property to form non-amyloid nanofibers at acidic and low-ionic strength conditions.These fibrous nanostructures of r Balcp19 k are very stable and do not disassemble in seawater.Moreover,the adhesive ability of r Balcp19 k nanofibers is much stronger than that of unassembled rBalcp19 k monomers,and it shows resistance to the adverse effects of alkaline and high-ionic strength seawater conditions.Taken together,these discoveries probably indicate that upon synthesized in the acidic cement gland,cp19 k self-assembles into nanofibers and barnacles secrete pre-assembled fibrous cp19 k nanostructures for strong underwater adhesion,adding new insights into the old model of barnacle underwater adhesion mechanisms.In addition,we discover that the intramolecular disulfide bond formed between the two conserved Cys residues of Balcp19 k is not essential for maintaining its secondary structures and adhesive abilities.Instead,the Cys-substituted mutant shows broader self-assembly conditions.In the end,we design 9 Balcp19k-inspired peptides based on its self-assembling ability and block copolymer-like sequence property.Fascinatingly,it is observed that multiple peptide mimics of Balcp19 k are able to self-assemble into fibril nanostructures with distinct morphologies.Especially,a peptide rich in “Ser-Ala” repeat and designed from the central region of Balcp19 k shows the unique capability to self-assemble into toroidal microrings.Moreover,these toroids that have not been observed before can be formed in a broad range of conditions,and their morphologies can be tuned by varying peptide concentrations and solution conditions.To summarize,our discoveries not only verify the self-assembly ability of rBalcp19 k,but also point to a new barnacle-inspired protein/peptide nanotechnology research direction.