Recombinant Expression Of Mussel Adhensive Proteins In Escherichia Coli And Bacillus Subtilis

Mussel adhesive proteins（MAPs）has the characteristics of strong adsorption and water resistance,and can be used as bioadhesives in the fields of bioengineering and biomedicine.These characteristics are mainly due to the 3,4-dihydroxy-phenylalanine（Dopa）in MAPs mediating a variety of interactions.However,the yield of MAPs directly extracted from mussels is extremely low,which hinders its application.Predecessors used prokaryotes,eukaryotes and other hosts to express MAPs,but the biggest problem of recombinant expression is the low modification rate of Dopa in MAPs and it is difficult to obtain strong adhesion.Therefore,there is an urgent need to improve the expression of MAPs and the efficiency of Dopa modification.Mussel foot protein MFP5 is the protein with the highest dopa content in MAPs,followed by MFP3.The main purpose of this study is to express Mussel foot protein（MFP）in Bacillus subtilis to increase the expression level,and to modify the tyrosine to Dopa in the recombinant protein.First,refer to previous methods to express and modify MFP3 and MFP5 in Escherichia coli^[113],and optimize the incorporation efficiency of Dopa;secondly,express MFP3 and MFP5 by plasmid vector and fusion in Bacillus subtilis.The results obtained are summarized as follows:1.Expression and modification of MFP in Escherichia coliIn this study,the intracellular expression of MFP3 and MFP5 was achieved in the strain BL21,and the tyrosine was converted into Dopa by incorporation of Dopa.Tyrosyl tRNA synthetase（TyrRS）can recognize Dopa,but its affinity for tyrosine is about 200 times higher than Dopa.In order to enable TyrRS to bind Dopa without using tyrosine,a tyrosine auxotrophic strain was first constructed.Secondly,use basal medium containing Dopa for culture when inducing protein expression.The detection of Dopa in MFP by NBT staining indicated that the tyrosine in the protein was at least partially converted into Dopa.In order to increase the incorporation rate of Dopa in MFP,the conditions of adding Dopa were optimized.The results showed that Dopa was added when the bacteria grew to OD600 of 1.5,and the MFP contained the largest amount of Dopa.Finally,the temperature and p H characteristics of MFP were explored.The results show that the recombinant MFP has acid and alkali resistance and can exist stably at 0-60℃.2.Intracellular expression of MFP in Bacillus subtilisBacillus subtilis has the characteristics of safety and strong secretion ability,so it is a commonly used host for expressing foreign proteins.In this study,the mfp gene was first integrated into the Bacillus subtilis chromosome,but the target protein was not detected by SDS-PAGE.Secondly,try fusion expression,but the expression level of fusion protein is low.In order to increase the expression of MFP,a multi-copy plasmid was used to induce the expression of MFP.Firstly the xylose promoter -35 region,-10 region and RBS are rationally modified,and use green fluorescent protein as the reporter gene to characterize the expression intensity.Then a stronger plasmid vector was selected to express MFP,and the intracellular expression of MFP3 and MFP5 was achieved in Bacillus subtilis.3.Secretory expression of MFP in Bacillus subtilisBacillus subtilis has a complete protein secretion system that can secrete a large amount of target protein into the medium,which not only improves the quality of the target protein,but also simplifies the downstream purification steps.In this study,the commonly used signal peptide SPapr E of Bacillus subtilis was selected to express MFP,but the target protein could not be detected by SDS-PAGE.In order to increase the extracellular expression of MFP,the well-secreted methyl parathion hydrolase（MPH）was used to fusion expression with MFP3 and MFP5.However,the amount of expression is significantly reduced after fusion,which is difficult to meet practical applications.Further attempts to fuse other secreted proteins,such as spheroplast protein Y（SPY）as a chaperone protein can help the target protein fold and increase the yield.Firstly,it was verified that SPY can be secreted and expressed in Bacillus subtilis and then the protein was expressed by fusion with MFP.The results show that both SPY fusion MFP3 and MFP5 can be secreted and expressed,and the secretion expression level of SPY fusion MFP is significantly higher than that of MPH fusion.In summary,this study expressed and modified MFP3 and MFP5 in E.coli.On this basis,the Dopa incorporation conditions were optimized to increase the Dopa incorporation rate.In Bacillus subtilis,firstly,the intracellular expression of MFP3 and MFP5 was realized by optimizing the plasmid expression;secondly,the secreted expression of MFP3 and MFP5 was realized by the fusion partner protein.The disadvantage is that the MFP has not been modified in Bacillus subtilis,and the modification method in E.coli can be used for subsequent research.