Preparation And Performance Studies Of Fusion Protein Adhesives Based On Marine Adhesive Proteins And Elastin-Like Polypeptides

Wet adhesion technology has potential applications in various fields,especially in the military and biomedical fields.In order to master this technology,researchers learn from nature and have been attempting to imitate the outstanding underwater adhesion ability of many biological adhesives secreted by marine organisms(such as mussels,sandcastle worms,and barnacles)to develop biomimetic underwater adhesives.However,to design biomimetic adhesives with more superior adhesive performance,there is an urgent need to integrate different adhesive strategies of different bioadhesives instead of only imtate one single adhesive mechanism.To engineering combinational biomimetic adhesives,in this paper,we selected elastin-like polypeptides(ELP)with high yield,easy purification procedure and liquid-liquid phase separation(LLPS)property,as well as Balanus albicostatus cp19k(Balcp19k)which can self-assemble into amyloid fibers and Mytilus edulis fp5(Mefp5)that depends on Dopa for adhesion as modules,to design a class of fusion protein adhesive based on marine adhesive proteins and ELP.(1)A novel expression vector named pET21a-ELP64 was first constructed by recursive directional ligation.Then,gene sequences of Balcp19 k and Mefp5 were inserted into pET21a-ELP64 and pET ELP110(a purchased commercial vector)via gene cloning.Through this way,we successfully constructed four different recombinant plasmids(pET21a-ELP64-Balcp19k/Mefp and pET ELP110-Balcp19k/Mefp5).(2)Heterologous expression of the four fusion proteins were carried out in Escherichia coli.Through gradually optimizing the expression conditions(including temperature,time and concentration of isopropyl-β-D-thiogalactoside),we successfully achieved efficient expression of ELP110-Balcp19 k and ELP110-Mefp5,and found that the former was better expressed at low temperature(17℃)while the latter was highly expressed at 37℃.(3)The fusion proteins ELP110-Balcp19 k and ELP110-Mefp5 were successfully purified by affinity chromatography.In the meanwhile,we developed a new fusion protein purification approach that combines high temperature-induced precipitation with inverse transition cycling(ITC).Fusion proteins were also purified with this approach.Compared to affinity chromatography,the new method has many advantages such as quick,low-cost,and more efficient.(4)Performance characterizations of ELP110-Balcp19 k adhesive found that it has the ability of LLPS and self-assembly into nanofibers,and that after LLPS and self-assembly the adhesive shows the strongest adhesive shear strength(It exceeds 4MPa on glass and steel in air and reaches to 0.31 MPa on glass in humid environments,whereas it is weak underwater and needs to be improved in the future).Moreover,the adhesive has the advantages of injectability and good biocompatibility,showing great potential in biomedical fields.This work exploits a barnacle cement protein as a self-assembly domain that forms nanofibers to fuse with ELP(a LLPS domain)to prepare recombinant protein adhesives for the first time.It proves the idea that combinational design can significantly improve the adhesion strength of biomimetic adhesives and provides new insights into the design of combinational biomimetic adhesives.