Expression And Functional Characterization Of The 52kDa Cement Protein (cp-52k) In Megabalanus Rosa

Barnacle,the most common marine biofouling organism,can firmly and permanently attach to the underwater surface via self-secreted multi-protein complex named barnacle cement.Obtaining the composition,structure and function of each protein in the barnacle cement is the key point to understand the adhesion and curing mechanism.It will provide new inspiration for the research of new technology of anti-fouling and biomimetic underwater stong adhesive.In this thesis,we selected the cement protein52kDa in Megabalanus Rosa(Mrcp-52k),which is the most abundant protein and plays vital role in cement curing.It is the first time to obtain the recombinant Mrcp-52k(rMrcp-52k)with functional activity by heterologous expression and carry out its function research.So that the components of barnacle bio-adhesive analysis and adhesion curing mechanism has taken a step forward.In view of the naturally isolated Mrcp-52k is a specific protein with strong hydrophobicity and glycosylation modification,it is difficult to express the active protein in the heterologous system.Based on the sequence of Mrcp-52k gene,which was deduced according to the way of dissolving curing cement by the research group of Kamino in 2012,the resulting gene was expressed in E.coli and Pichia Pastoris.In accordance with codon bias in E.coli and Pichia Pastoris,the gene sequences of Mrcp-52k were chemical synthesized,and successfully constructed into expression vectors,including pET-32a(+),pET-21a(+)and pPICZ?A,respectively.Mrcp-52k gene was highly expressed in E.coli BL21(DE3)under the induction condition of 27?,and high purity rMrcp-52k was obtained by affinity chromatography.Coomassie brilliant blue(CCB)staining and quartz crystal microbalance(QCM)were applied to qualitatively and quantitatively characterize the adhesion ability of rMrcp-52k,respectively.The results showed that rMrcp-52k possessed the same adhesion properties as the commercialized Cell-Tak~?.The results of scanning electron microscope(SEM)showed that the dense grid fibers were formed on the surface,and this structure was favorable for the adhesion of the protein on the surface of the material.In addition,rMrcp-52k was stained with thioflavin T(ThT)for amyloid fibril detection and observed by atomic force microscopy(AFM).It was found that rMrcp-52k formed self-assembled amyloid fibrils.