Rational Design And Thermal Stability Of Cu, Zn-SOD

Superoxide dismutase(SOD)is also known as liver protein.It is the specific scavenger of the superoxide anion free radical(·2O),which can prevent and treat a variety of diseases caused by superoxide anion such as rheumatoid arthritis,emphysema,senile cataracts and so on.Currently,in addition to be applied to clinical,SOD has also been added to a variety of food and cosmetics as a health care products and anti-aging.So,SOD is a promising protease.However,due to natural SOD in the body has the shortcomings such as a short stagnation period,unstable,immunogenicity,easy to protease hydrolysis and so on,thus limiting the large-scale production and extensive application of it.Therefore,to solve the protein defects,design and development of high activity,strong stability of the new SOD is very necessary.In this study,the amino acid sequence of Cu,Zn-SOD protein in Mus Musculus of NCBI database was simulated by Discovery Studio 4.0 software,and molecular dynamics simulation was carried out and determining the mutation site according to the energy change,the final mutant amino acid was obtained by molecular dynamics simulation.Finally,the recombinant plasmids of WT-SOD and MSOD were constructed and cloned into Escherichia coli DH5? and BL21(DE3)for cloning and expression.The thermal stability of Cu,Zn-SOD and MCu,Zn-SOD was verified by experiment.The process and results of this subject are as follows:1.The amino acid sequences of Cu,Zn-SOD from Human,Bos Taurus,Pongo abelii,Monkey,Mus Musculus were received from NCBI.The amino acid sequence of the above amino acid sequence was matched with Mus Musculus sequence by software ClustalX2,and we got the conserved amino acid sequence of Mus Musculus by DNAman analyzing.The corresponding 27 unconserved amino acid sequences were used as the sites to be mutated.2.The crystal structure of Cu,Zn-SOD molecules of Mus Musculus was obtained from the Protein Data Bank database by accession number 3GTT and loaded into the software Discovery Studio 4.0,The unconserved amino acids to be mutated in Cu,Zn-SOD were mutated into 19 other amino acids by virtual amino acid site-directed mutagenesis.We finally got the best TOP5 mutants of Single-MSOD,Double-MSOD,Triple-MSOD based on the energy change.3.We carried 10 ns molecular dnamics assays of the best TOP5 mutants of S-MSOD,D-MSOD,T-MSOD;finally we get the RMSD,RMSF,Potential energy,H-Bond by the analysis trajectory model.According to data trends,the best mutated combinations are:Single Mutation(Q49W);Double Mutations(Q49W/G90I);Triple Mutations(V30W/Q49W/G90I).4.We got the gene from the blood of the Kunming mouse,and the recombinant plasmidspGEX6p-1-SOD,pGEX6p-1-S-MSOD,pGEX6p-1-D-MSOD,pGEX6p-1-T-MSODwere prepared by PCR,Overlap PCR,digestion,ligation and transformation.5.The recombinant plasmids were transferred into BL21(DE3)for induction expression,SDS-PAGE electrophoresis was performed on the whole bacterial liquid,supernatant and precipitate after repeated freezing and thawing.It was confirmed that Cu,Zn-SOD produced specific protein bands at about 16 kD in the supernatant,which indicated that Cu,Zn-SOD had appropriate soluble expression.GST-SOD and GST-MSOD were obtained by GST agarose as the medium by affinity chromatography.Thrombin cleaves the fusion protein to obtain pured SOD and MSOD.6.The activity of SOD and M-SOD were measured by pyrogallol method.The activity of WT-SOD was 714 U/mg;The activity of S-MSOD was 839 U/mg;The activity of D-MSOD was632 U/mg;The activity of T-MSOD was 868 U/mg.The activity of S-MSOD was 17.5% higher than that of WT-SOD,T-MSOD was 21.6% higher than that of WT-SOD,and D-MSOD was13% lower than that of WT-SOD.7.The stability of WT-SOD and MSOD was determined by pyrogallol method also.Finally,when the enzymes were placed at 85? for 1 h,the relative activity of wild type protein was about 10%,the S-M-SOD was 20%,the D-MSOD was 5%,the T-MSOD was 35%.