A Study On Construction,Nature And Function Of The Recombinant Cement Protein Cp20K In Balanus Albicostatus

Barnacles rely on their secreted bioglue to achieve excellent underwater adhesion,however,the functional mechanisms of the proteins in the bioglue remains unclear.In this paper,cp20k,as a major cement protein in barnacle cement,was studied.After obtaining recombinant cp20k and its mutants,its self-assembly properties,calcite-specific adsorption function and interaction with other cement proteins were studied to further understand its functional mechanisms in the curing of the bioglue.For better understanding of the effect of the conserved cysteine on the function of cp20k,it is necessary to obtain cp20k close to the native sequence and its mutants.Hence,based on the analysis of the primary structural characteristics of cp20k of Balanus albicostatus?Balcp20k?,two mutants that was important in the disulfide formation were designed,and we solved the inclusion body problems in the expression process by regulating the purification methods,etc.,to obtain the recombinant Balcp20k?rBalcp20k?and its mutants.The self-assembly properties of cp20k were studied.The aggregation behavior of rBalcp20k was triggered by the change of p H,which might be self-assembly.At approximate ocean pH?pH8.0?,rBalcp20k aggregates to form water-insoluble precipitated material.Compared with the mutants,and the disulfide bond breaking can enhance the solubility of this precipitate and alter its microscopic morphology.For the first time,the interaction between cp20k and other cement proteins was firstly detected.rBalcp20k interact with rBalcp19k only at pH8.0.That may suggest that the transition from the cement gland to the marine environment is a trigger factor for the aggregation of cp19k and cp20k.In addition,the effect of glycosylation of cp52k on the cross-linking with cp20k was investigated by using some carbohydrates such as lactose as substitutes for cp52k.The results showed that glycosylation of cp52k might not take part in the cross-linking of cement proteins.In view of the current understanding of"the main function of cp20k is to adsorb the calcium-base plate of the adult barnacle?the main component is calcite?",we verified that rBalcp20k can adsorb calcite,confirmed the interaction between the rBalcp20k and Ca²⁺,and discovered that the interaction between the mutants and Ca²⁺drops to a certain extent.Therefore,we propose that the disulfide bonds of cp20k benefit Ca²⁺binding,which may support that cp20k help biomineralization of the barnacle base plate.