Study On The Protein Components And Curing Mechanisms Of The Balanus Reticulatus Cement

Barnacles are a kind of typical marine fouling creatures that attach themselves robustly to almost any underwater substrates through multiple protein-based "barnacle cement".Barnacle cement has drawn considerable attention due to its’ excellent wet adhesive property and severe fouling issues.Thus,it’s of great importance to deeply reveal the characteristics of cement proteins and to illustrate the curing mechanisms,which would inspire barnacle-based bionic design and targeted anti-fouling strategies.We extracted cement proteins of Balanus reticulatus that is widely distributed in China near-shore and then identified them by N-terminal sequencing and mass spectrometry based on the transcriptome database.What’s more,we assessed the spontaneous amyloid fibril aggregation behavior of Balcp19 k in seawater to explain the mechanism of curing process.The main work and conclusions of this paper are as follows:The cement was partially rendered soluble by a high concentration of denaturant,chemical cleavage reagent or amyloid fibrils deaggregation reagent.Finally,more than 7 bands could be seen after SDS-PAGE.A major protein that has an apparent molecular of about 40 kDa was confirmed to be a novel cement protein by mass spectrum based on the NCBIprot and Swissprot database.We further purified the protein by HPLC and then performed the Edman N-terminal sequencing,which failed to acquire any amino sequence information due to the N-terminal closure.In order to solve the main problems including no available genomic information,difficulty to enrich the low abundance protein and N-terminal closure,in this paper,the transcriptome of the main body soft tissue of the Balanus reticulatus was sequenced by high throughput RNA-seq to serve as the reference database for mass spectrometry identification.Through tandem mass spectrometry analysis based on the transcriptome database,few homologous proteins that are already reported in other barnacles,were successfully identified.At the same time,a novel protein,coded by the transcript c94902_g3₂,with a theoretical molecular weight of about 46 kDa was found.Utilizing the pET21a-Balcp19 k plasmid that was already constructed before by our laboratory the Balcp19 k protein was successfully expressed in E.coli BL21（DE3）and purified by combinational methods of metal chelate affinity chromatography and reverse phase chromatography.The final product was validated by MALDI-TOF-TOFand Western-blot.We then assessed the behavior self-assembling into amyloid fibrils of the Balcp19 k using thioflavin T staining,micromorphology analysis and secondary structure analysis.Further measurement of the self-assembly kinetic curve and solubility analysis of the self-assembled product suggested that the process of amyloid fibrils aggregation in seawater was closely related to the curing procedure.In this paper,we identified few homologous proteins of several known cement proteins and found a new protein.In addition,we for the first time reported the spontaneous amyloid fibrils aggregation of an intact cement protein in seawater.The results of the study are of great significance to further explain the wet adhesion mechanisms of barnacle cement and conductive to biomimetic design and antifouling materials development.