Recombinant Expression Of Shrimp Lysozyme In Escherichia Coli And Refolding Of Shrimp Lysozyme Inclusion Bodies

In China,the shrimp Fenneropenaeus is very popular to customers because it can provide a high nutritional value.But some diseases severely influenced the development of aquaculture shrimp in the last decade.In order to solve the problems,many studies have been done.It has demonstrated that it is important to enhance the immunology mechanism of shrimp.Lysozyme is an important factor in shrimp immune system.It could destroy and eliminate the invasive pathogens in vivo by the way of hydrolytic process.As a protein of non-toxic,harmless and safe additives,lysozyme is more and more widely used in medicine,food and biotechnology.Lysozyme extracted from shrimp tissues is difficult to achieve industrial production for the low yield.The research has showed that recombinant shrimp lysozyme was highly expressed in prokaryotic expression system,but it mainly exist in the form of insoluble inclusion bodies.Therefore,it is useful to refold the inclusion and effective way to solve the contradiction of supply and demand.In this project,the FcLys(F.chinensis lysozyme)gene that was preserved in the cloning vector of pMD-18-T was connected to the expression vector of pET-30a(+)so as to construct the recombinant plasmid of pET-30a(+)-FcLys.Then it was transformed into E.coli Rosetta(DE3).The following step was to induce by IPTG and express the recombinant proteins of FcLys.The factors associated with the lysozyme solubility,such as temperature,concentration of IPTG and medium component were optimized.The inclusion body was washed twice with washing buffer and dissolved in the extraction buffer.Then the optimal conditions of dissolution was confirmed.The assays of dilution refolding and affinity chromatography refolding were applied to obtain the bioactive and soluble FcLys protein.The antibacterial activity of the refolded recombinant proteins of FcLys was detected according to agarose radial diffusion assay.The results showed that the recombine ant protein of FcLys was highly expressed in E.coli Rosetta(DE3)as intracellular insoluble inclusion bodies.With the variation of temperature,concentration of IPTG and medium components could not increase solubility of FcLys.The optimal dissolving conditions was that extraction buffer containing(3-mercaptoethanol dissolved inclusion body at 30? for 2 h.To dilution refolding,the yield of refolding increased when the FcLys protein witih lower concentration was gradiently refolded under 2 mol/L urea at pH 8.0.To affinity chromatography refolding,it demonstrated that the yield of refolding increased when the FcLys sample with lower concentration was loaded to the column under higher temperature at pH 8.0.The product from refolding showed that the FcLys protein possesses significant activities against Gram-positive bacteria Microoccus lysodeiki?us and Staphyloccocusaureus.So they can be widely used in aquaculture,livestock and poultry breeding,feed additives and food processing industry.Therefore,the products in this study have great market potential and development value.