Construction Of Barnacle-derived Adhesive Protein Based On Fused Functional Module

Compared with chemically synthetic adhesives,barnacle-derived protein adhesives have the advantages of environmental friendliness and underwater adhesion,which makes it a hot spot in the research and development of biomimetic adhesives.In this study,Megabalanus rosa protein Cp19k（Mrcp19k）was chosen as the viscous component of the protein adhesive.In order to improve the underwater viscoelasticity and the adhesion performance in non-aqueous environments of Mrcp19k,genetic engineering method was used to construct various barnacle-derived fusion adhesive proteins with different protein functional modules.The adhesion function of adhesive protein was characterized.The main research contents and results of this paper are as follows:1.Using heterogeneous charge function module of Ddx4 to promote the aggregation of protein,the fusion adhesiv protein Mrcp19k-Ddx4 was constructed.The underwater viscoelastic properties of the adhesive protein was tested with quartz crystal microbalance with dissipation（QCM-D）.By comparing the crystal frequency changes caused by the adhesive protein（Δf,reflecting the effect of the adhesive protein adhesion ability）and crystal dissipation change（ΔD,reflecting the elastic ability of adhesive protein）,it was found that Mrcp19k-Ddx4 could increaseΔf from 22.25 Hz of Mrcp19k to 32.50 Hz,and increasesΔD from 1.18×10^-6u of Mrcp19k to 1.53×10^-6u,which indicated that the fusion adhesive protein Mrcp19k-Ddx4 had better underwater viscoelastic properties than the non-fusion adhesive protein Mrcp19k.The adhesion test in a non-aqueous environment indicated that Mrcp19k-Ddx4 could increase the adhesion strength of Mrcp19k from 0.85MPa to 1.01 MPa.The experimental results show that functional module Ddx4 could improve the underwater viscoelastic properties of the adhesive protein and the adhesion performance in non-aqueous environments of adhesive protein Mrcp19k.2.Using the protein functional module SpyCatcher/SpyTag which can form isopeptide bonds and the elastic functional module elastin-like polypeptide（ELP）,the fusion protein Spy Catcher-ELP-Mrcp19k-ELP-Spy Catcher（CC-Mrcp19k）,Spy Tag-ELP-Spy Tag-ELP-Spy Tag（TTT）were constructed.Mixing fusion protein CC-Mrcp19k and TTT could form reticulated adhesive protein according to the molar ratio of Spy Catcher:Spy Tag=1:1 in the protein.The underwater viscoelasticity test experiment showed that theΔf of reticulated adhesive protein can increase from 22.25 Hz to 40.03 Hz,and theΔD of can increase from 1.18×10^-6u to 3.25×10^-6u,indicating Reticulum adhesive protein has better underwater viscoelastic properties than adhesive protein Mrcp19k.In the non-aqueous environment,the adhesion strength of reticulated adhesive protein can increase from 0.85 MPa to 1.26 MPa.The experimental results showed that the network structure formed by functional modules SpyCatcher/SpyTag and ELP could further improve the underwater viscoelastic properties of the adhesive protein Mrcp19k and the adhesion performance in non-aqueous environments of Mrcp19k.3.Using the functional module SpyCatcher/SpyTag which can form isopeptide bond,the circular adhesive protein Spy Catcher-Mrcp19k-Spy Tag（CT-Mrcp19k）was constructed to reaserch how circular structure influence the adhensive function of adhensive protein Mrcp19k.Comparing to Mrcp19k,theΔf of reticulated adhesive protein can increase from 22.25 Hz to 25.72 Hz,and theΔD increase from 1.18×10^-6u to1.42×10^-6u,which indicated reticulum adhesive protein had better underwater viscoelastic properties than adhesive protein than Mrcp19k.The adhesion test in a non-aqueous environment showed that the adhesion strength of reticulated adhesive protein reached1.81 MPa,which was 1.97 times that of the adhesive protein Mrcp19k.The experimental results show that reticulated structure formed by the functional module Spy Catcher/Spy Tag could improve the underwater viscoelastic performance of Mrcp19k,and further improve the adhesion performance of Mrcp19k in a non-aqueous environment.