The Optimization Of Recombinant Expression System And Study On Mechanism Responsible For The Stability Of MBSP From Crucian Carp

Enzemy engineering plays an increasingly important role in the food,medicine and chemical industry.However,most of the enzymes have poor tolerance towards extreme environments in real industrial applications,which greatly limits their applications.Thus,it is critical and chal enging to improve the stability of enzymes.Therefore,study on mechanism responsible for the stability of myofibril-bound serine proteinase(MBSP)with high stability from crucian carp will lay the foundation for other enzymes.In this study,we have successful y constructed the prokaryotic expression strain and obtained recombinant MBSP(rMBSP),which exists in the form of inclusion body,and then polyclonal antibody of anti-MBSP was obtained from immunized rabbit.Then,the suitabe Pichia pastoris expression system for MBSP was established by optimizing expression vector,host and expression conditions.Finally,site-directed mutagenesis and recombinant expression of MBSP were realized using bioinformatics analysis,overlap extension PCR and gene recombination techniques.The related residues with stability of MBSP have been confirmed by comparing enzymatic properties between wild type MBSP and its mutants,and the mechanism responsible for the stability of MBSP was analyzed through comparative of structure models of MBSP and its mutants.The prokaryotic expression strain Rosseta/pET-28a-MBSP was successful y constructed using gene recombination technique,and it was induced to express rMBSP in the form of inclusion body using lactose as inductor.The rMBSP was purified by Ni-NTA agarose affinity column chromatography,and then using it as the antigen to immune the rabbit,and the specificity polyclonal antibody of anti-MBSP was obtained from immunized rabbit.In order to obtain rMBSP with the biology acitivity,we constructed four Pichia pastoris expression system,GS115/pPIC9K-MBSP,SMD1168/pPIC9K-MBSP,GS115/pPICZ?A-MBSP and SMD1168/pPICZ?A-MBSP.Western blot and MALDI-TOF-MS analysis showed that the molecular weight of the rMBSPs were approximately 28 kDa,33 kDa and 36 kDa respectively.The characterization of rMBSPs were investigated,and the results showed that 28 kDa rMBSP from GS115/pPICZ?A-MBSP and SMD1168/ pPICZ?A-MBSP was similar with native MBSP.Because of its less background proteins,the SMD1168/pPICZ?A-MBSP was chosenas the suitabe Pichia pastoris expression system for MBSP.The maximum expression level was obtained under the pre-induction optical density OD600=5.0,1% methanol concentration,pH=6.5 and induction time 96 h.The related residues with stability of MBSP were predicted by alignment with primary structure and tertiary structure of crucian carp MBSP and trypsin using bioinformatics tools,and they were identified by the site-directed mutagenesis and gene recombinant expression.The results showed that the mutant P95 T exhibited decrease both half-life for thermal inactivation at at 65? and themal stability,which demonstrated that Pro95 is key amino acid residue to maintain the high themal stability of MBSP;the mutant R125 N exhibited increase pH stability,which showed that Arg125 is key amino acid residue for pH stability of MBSP;the mutants A127 S,I130D and A127S/I130 D improved the themostablity,pH stability and chemical denaturation of MBSP,and the mutant A127S/I130 D showed a greater improvement in stability than thier single mutants,which demonstrated that Ala127 and Ile130 is key amino acid residue to influence the stability of MBSP and have synergistic between them.The mechanism responsible for the stability of MBSP was analyzed through comparative of structure models of MBSP and its mutants.