| Mussel foot proteins(MFPs)is a type of compound proteins cluster secreted by marine mussels' foot glands that can firmly adhere to the surface of various materials in aquatic environment,such as reefs and ship hulls.They have high strength and toughness,strong water resistance and substrate adhesion property,good biocompatibility and degradability.These significant advantages render them to have a broad and irreplaceable application prospects in the fields of medicine,marine engineering,organic coatings and so on.Initially,mussel foot protein was extracted from natural mussels via chemical method.The extremely low yield and high cost make it difficult to meet industrial demands.Then,a variety of mussel glue viscous water Gels were obtained via synthetic methods.But the poor biocompatibility greatly limited their application.Recently,researcgers have begun to use genetic engineering methods to produce mussel viscous protein and have made certain progress,but it is still difficult to produce mussel viscous protein on large scale.Therefore,in this thesis,based on previous researches and the special requirement of engineering,a variety of engineered strains were constructed in the prokaryotic system through genetic engineering to express mussel foot proteins and their fusion proteins.The amino acid residues of mussel viscous proteins were modified via in vivo and in vitro oxidation,and improved thier adhesion performance to meet the engineering requirement.The main research work and results are summarized below:1.Taken the tyrosine-rich Mytilus galloprovincialis foot protein type 5(Mgfp-5)as the core,the fusion genes were designed by using genetic engineering technology,finally constructed four mussel protein gene recombination plasmids p ET28a-mgfp-5,p ET28a-mgfp-151,p ET28a-mgfp-5-lc and p ET28a-mgfp-151-lc to obtain a series of prokaryotic expression strains.The optimal fermentation conditions were at OD600?0.8,the induction concentration of IPTG at 1 m M,37 ? for 6?8 h,,the protein expression was relatively high;For the four mussel adhensive proteins M5,M151,5LC,151 LC,their corresponding crude protein yields were about 170.55 mg/L,193.47 mg/L,203.10 mg/L and 200.80 mg/L.Then,the crushed supernatant was collected,purified by a Ni column,and eluted with 8 M urea to obtain pure mussel viscous protein solutions.The corresponding purification rates were 12.59%,11.97%,12.83%,and 14.46%.2.GFP and RFP were used as reporter proteins to verify the feasibility of co-expression of p ET28 a and p ACYCDuet1 in E.coli.On this basis,the mussel viscous protein gene and the tyrosinase gene were respectively cloned into the above vectors,and the mussel viscous protein in vivo oxidation system was successfully constructed,and realized the in vivo oxidation of the above four mussel viscous proteins..Taking the DOPA content in the original mussel viscous proteins as the starting point,the DOPA content in the four mussel viscous proteins M5,M151,5LC and 151 LC in the co-expression system were increased by 4.4,3.3,5.0,and 5.3 times,respectively.3.Another tyrosinase engineered bacteria was constructed,and the conditions of tyrosinase oxidation in vitro were also explored.It was found that when the substrate concentration was 2 m M and the p H was 4?7,the obtained tyrosinase showed a relatively stable performance.The oxidation effect from tyrosine to DOPA could reach the maximum oxidation rate(45%)within 4 h.Under these conditions,the mussel viscous protein was oxidized in vitro,and the results showed that the DOPA content of the four mussel viscous proteins M5,M151,5LC and 151 LC,were respectively increased by 7.9,8.9,12.0 and 10.8 folds;while the DOPA content of the four mussel mucins that underwent double oxidation in vivo and in vitro were increased by 20.8,22.7,21.0 and22.1 times,respectively,which is 4 to 5 times that of only in vivo oxidation.At the same time,the tyrosine conversion rates of the four mussel mucin proteins after double oxidation in vivo and in vitro reached 9.06%,11.76%,11.80% and 10.57%,respectively.4.The adhesion performance test of the mussel viscous proteins.The purified mussel viscous protein eluate was renatured by dialysis with a gradient concentration of urea solution,and then freeze-dried to obtain pure protein powder under different treatments.The adhesion performance test was carried out with a universal testing machine.The results showed that the double oxidation in vivo and in vitro could increase the adhesion performance of mussel viscous proteins by an average of about 5 times,and the maximum adhesion strength could reach 0.74 MPa.At the same time,it also shows that the adhesion performance of mussel viscous protein is obviously positively correlated with the content of DOPA in mussel viscous proteins. |
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