Development Of Biomimetic Adhesive Peptides Based On Mussel And Sandcastle Worm

The marine biomimetic adhesive materials are widely applied in many fields such as medicine,chemical engineering,and national defense construction.The performance of adhesive materials is determined by both interfacial adhesion and cohesion.Many studies indicate that both mussel foot proteins（Mfps）and cement precursor proteins of the sandcastle worm phragmatopoma californica（Pcs）achieve high interfacial adhesion through 3,4-dihydroxyphenylalanine（Dopa）.In addition,Pcs can be classified into polyanionic peptides and polycationic peptides,which achieve high cohesion through electrostatic-mediated complex cohesion.Inspired by mussels and sandcastle worms,our study proposed a new strategy for achieving interfacial adhesion based on Dopa and achieving cohesion based on electrostatic binding of anionic and cationic peptides.Through the strategy,novel biomimetic adhesive peptides were designed and synthesized,laying the foundation for the development of new biomimetic adhesive materials.The natural mucins containing Dopa in Mfps and Pcs was used as candidate anionic and cationic peptides based on the above strategy to design novel bionic adhesion materials.Specifically,the negatively charged natural protein Pc3a1 was used as an anionic peptide containing Dopa,and the Mfp5-Mfp3-Mfp5 and Pc1-Mfp5 fusion proteins were constructed by concatenating positively charged natural proteins Mfp3,Mfp5,and Pc1 as cationic peptides containing Dopa,both of which were combined by electrostatic force to form a candidate bionic adhesive material.The soluble expressions of the above anionic and cationic recombinant proteins were achieved by the insect expression system,which not only had a relatively short expression cycle,but also avoided the formation of inclusion bodies.In details,the recombinant baculovirus plasmid（Bacmid）was generated by transposing the target genes into the baculovirus genome using p Fast Bac HBM-TOPO vector and DH10Bac competent cells.Then the recombinant Bacmid was further transfected into insect cells to prepare the baculovirus,by which the soluble expression of the target protein was achieved.The results of agarose gel electrophoresis analysis and sequencing confirmed that the recombinant Bacmid containing target genes were obtained.The flow cytometry results showed that baculovirus containing the target genes were generated with concentrated titers of8.87×10⁸ ivp/m L,1.13×10⁹ ivp/m L and 1.08×10⁹ ivp/m L,respectively.The results of WB and SDS-PAGE verified that the fusion proteins Mfp5-Mfp3-Mfp5 and Pc1-Mfp5were expressed.The Multiplicity Of Infection（MOI）as well as the length of infection were subsequently optimized,but the low total expression and the high proportion of heterologous proteins hindered the purification and preparation of target proteins.At the same time,through further analysis of the amino acid sequence characteristics and action mechanism of Mfps and Pcs,the anionic and cationic peptides with adhesion and composite cohesion were also designed.Specifically,the natural proteins with good adhesion properties,Mfp5 and Pc3a1,were selected as templates,one anionic peptide and three cationic peptides were designed according to relevant studies.The above polypeptides were synthesized by solid phase peptide synthesis（SPPS）technology.The SPPS technology can precisely synthesize and modify the target polypeptides,and has the advantage of high product purity.The MS and HPLC results confirmed that the target anionic and cationic peptides were synthesized with a purity of over 95%.The tensile strength test results showed that the adhesion pressure of the anionic peptides and three cationic peptides on the surface of Si O₂ reached174.04 k Pa,166.25 k Pa,180.11 k Pa,and 158.87 k Pa,respectively.After mixing with the anionic peptides,the adhesion pressure of the three cationic peptides increased to280.27 k Pa,264.81 k Pa,and 241.15 k Pa,respectively.The AFM test results showed that the adhesive forces at the nano-scale of the anionic peptides and the two cationic peptides with relatively better adhesive properties were 4.95 n N,9.24 n N,and 5.43 n N,respectively.After mixing with the anionic peptides,the adhesive forces at the nano-scale of these two cationic peptide mixtures increased to 18.54 n N and 15.82 n N,respectively.Furthermore,the QCM test results showed that the adsorption quantities of the above two cationic peptides on the Aurum（Au）surface were 82.69 ng/cm² and265.13 ng/cm²,respectively,which were increased to 151.53 ng/cm² and 320.95 ng/cm²,respectively,by mixing with anionic peptides.The above research results indicated that the adhesive peptides designed based on the new strategy had good adhesive properties,and suggested that electrostatic-mediated complex cohesion could significantly enhance the adhesive properties.In summary,we proposed a new strategy for designing biomimetic adhesive materials,based on which novel adhesive peptides with good performance were designed and synthesized.Therefore,this study provides a reference for the development of novel bionic adhesive materials,which is of great scientific significance and application value.