Functional Study Of The Disulfide Bridges In The Self-assembly Of Hydrophobin

Hydrophobins are amphiphilic proteins of small molecular weight produced by filamentous fungi.Hydrophobins have been found to have extremely high surface activity by self-assembly on the surface to form amphiphilic protein membranes,thereby changing properties of the interface.In recent years,based on the self-assembly properties of fungal hydrophobins,hydrophobins have been applied in various fields,including biosensors,drug carriers,emulsification,protein purification tags,enzyme immobilization and functionalization of two-dimensional materials.In order to further study different hydrophobins,hydrophobins are classified into type I and type II according to their physical and chemical characteristics and the degree of solubility of the protein membrane formed by self-assembly.Interestingly,all hydrophobins,either type I or type II,contain eight conserved cysteines,forming four intramolecular disulfide bonds.It can be seen that the self-assembly of hydrophobins and the conservative presence of disulfide bonds are extremely important for the function of hydrophobins.However,the effect of disulfide bond formation on the self-assembly of hydrophobin is largely unknown.In this paper,we use E.coli as the expression system,and design to mutate cysteine in type I hydrophobin HGFI and type II hydrophobin HFBI respectively.Through construction of vector,protein expression and protein isolation and purification,the protein were highly expressed and showed as a highly soluble protein.The subsequent tests of circular dichroism,quartz crystal microbalance,contact angle,ThT fluorescent dye,Congo red adsorption and emulsification showed that disulfide bonds have different effects on the self-assembly of hydrophobin.They do not participate in the self-assembly of the class I hydrophobin HGFI but directly affect the self-assembly of the class II hydrophobin HFBI.And fungal expression systems(such as Pichia)are mostly used for the current production of hydrophobins,but the use of fungal expression systems makes fungal cycles long because of the complicate steps such as methanol induction.For the production of the hydrophobin silk,the growth cycle is up to three weeks and mycelium yield is lower.For the prokaryotic system(such as E.coli)is simple to operate with high expression,but in the expression of wild-type hydrophobin,protein folding modification process makes the formation of inclusion bodies,which in turn involves the inclusion of denatured and refolding operations,which greatly increases the production cost and the production cycle,making it difficult to mass-produce and purify hydrophobin.We found that self-assembled cysteine mutants efficiently disperse carbon nanotube and graphene like natural hydrophobins,demonstrating the great potential for developing new applications based on hydrophobin cysteine mutants.It provides a new way for the purification and application of hydrophobin in the future.