The Study Of Mussel-inspired Antifouling Protein Coating

Biofouling caused by the attachment of protein and microorganism upon substrate surfaces has adverse impact on biomedical applications,as it may cause foreign-body reaction and in-stent restenosis and so on.The most effective strategy to eliminate biofouling is to construct anti-biofouling surfaces.However,chemical reactions were usually used for antifouling surface modification with complex procedures.Therefore,developing a facile surface modification strategy with biocompatibility is highly desirable.Inspired by the universal adhesive characteristic of marine mussel,we designed and exploited a recombinant mussel chimeric protein with a zwitterionic peptide(poly KE,K(lysine),E(glutamic acid))through synthetic biology strategy.The convertion of 3,4-dihydroxyphenylalanine(Dopa)was explored with in virto mushroom tyrosinase hydroxylation or in vivo post-translational modification with co-expressed tyrosinase.We also investigated the coating formation and antifouling performance of the chimeric protein.The gene of mussel adhesive protein and zwitterionic peptide were fused to construct the recombinant vector p ET-28a-MKE and then expressed in Escherichia coli(E.coli)at 37℃.After purification with 50%acetic acid and dialysis in 5%acetic acid,chimeric protein fp-MKE was obtained with the purity of 95%.Tyrosine residues were modified with mushroom tyrosinase in virto with a coversion rate of11.36%to prepare Dopa-containing chimeric protein mfp-MKE.After dip-coating on glass surface,mfp-MKE formed homogeneous and continuous protein coating.The results of antifouling experiments demonstrated that 2 mg/m L mfp-MKE protein coating could repulse more than 90%biopollutants compared with control.In addation,the hemolysis rates of protein coating were lower than 0.1%and the viabilities of NIH-3T3 after co-incubation were more than 99%,showing protein coating had excellent biocompatibility.To improve the conversion efficiency of tyrosine residues into Dopa,an in vivo tyrosine modification system was introduced into the recombinant protein expression system with both p ET-28a-MKE and p ACYC-dute-1-TYR in E.coli for post-translational modification of tyrosine in chimeric protein.The fermentation temperature was maintained at 16°C and cofp-MKE was purified through affinity chromatography with the purity of 70%.The results showed that the conversion efficiency of Dopa in vivo was 59.09%,which was higer than that in virto.Chimeric protein could form continuous coating on titanium,polystyrene and glass plates.The result of quartz crystal monitor measurement indicated that 0.25 mg/m L cofp-MKE protein coating could reduce 97.6%bull serum albumin adsorption to 10±0.3 ng/cm~2,and the coating could reduce more than 80%bacterial and cell adhesion.In conclusion,the prepared mussel-inspired recombinant chimeric protein with synthetic biology could readily form protein coating on various substrate surfaces with excellent fouling-resistant property.Taking advantage of mussel-inspired chimeric protein,a multifunctional surface modification platform can be developed through changing the incorporated peptide sequence,thus providing new insight into mussel-inspired surface modification strategy with potential applications in biomedical fields.