Virtual Mining And Functional Characterization Of Adhesive Proteins Based On Mussel Adhesion Mechanism

The adhesive protein secreted by the marine mussel byssus is the main means of its adhesion to the reef.Due to its favorable biocompatibility,mussel adhesive protein has been widely used in the field of bio-cement.Based on the main mechanism of mussel adhesive protein Mfps,tyrosine residues was oxidized by the tyrosinase to generate active dopa,Mfp3 and Mfp5,which play a major role in interfacial adhesion,were selected to carry out the following studies:(1)Mfp3 and Mfp5 tyrosine-rich polypeptides were selected for heterologous expression and purification in E.coli.According to the reported tyrosinase sequence,five homologous proteins were chose for expression and purification in E.coli,and recombinant tyrosinase with superior activity to the commercial enzyme was obtained.The highly active tyrosinase was applied to oxidize the tyrosine-rich peptides,and the oxidized gel-like product was tested on a universal testing machine to verify the adhesion of the tyrosine-rich peptides after oxidation.(The adhesive strength was 0.069 ± 0.019 MPa).(2)In order to obtain adhesive proteins with better adhesion performance,bioinformatics method was used for virtual mining of potential adhesive proteins from NR database.Having used a python script to perform the virtual mining of mussel-like adhesive protein sequence in the NR database,and a potential adhesive protein W1 sequence was obtained.The results of adhesion testing showed that the average adhesion strength of W1 was 0.07 ± 0.025 MPa,slightly lower than that of natural mussel Mfp3(0.085 ± 0.024 MPa).Due to the synergistic role of basic amino acids in the interface adhesion,the parameters of virtual mining were optimized,the adhesive protein 30 Y and 28 Y rich in tyrosine and basic amino acids was obtained,and the adhesion strength of 30 Y could reach 0.12 ± 0.019 MPa.Further freeze-dried treatment to the broken supernatant of the protein obtained a protein sample shaped like a liquid glue,and the highest adhesive strength of 28 Y was 0.48 MPa,which was about 5.6 times the measured adhesive strength of natural mussel Mfp3.In this study,bioinformatics was used for virtual mining of potential adhesive proteins,and its adhesive strength was significantly improved,which provided a novel strategy for the development of new bio-cement.