TAT-fusion Expression And Functional Activities Analysis Of Metallothionein From Freshwater Crab(Sinopotamon Henanense)

Metallothionein(MTs)are a class of cysteine-rich metal-binding proteins that can reduce free radical damage to the body.It plays an important role in anti-aging,anti-oxidation,and anti-tumor processes.In the early stage,the full-length MT c DNA was successfully cloned from the the freshwater crab(Sinopotamon henanense).In this study,human immunodeficiency virus transactivator(TAT)transmembrane peptide was introduced to analyze the physical and chemical properties of TAT-MT and the change of protein structure.Mmunofluorescence was used to detect the transmembrane activity of the recombinant fusion protein TAT-MT.The hydroxyl radical scavenging rate and total antioxidant capacity of TAT-MT was determined in vitro.MTT was used to determine the protective effect of recombinant protein on oxidative damage in cells.The results shown that:1.Bioinformatics analysis of TAT-MT recombinant fusion protein from the freshwater crab Sinopotamon honanenseTAT transmembrane peptide has transmembrane activity.the hydrophobic region and protein stability of TAT-MT are reduced,hydrophilicity?polarity and the isoelectric point of TAT-MT are increased.2.Construction of prokaryotic expression vectors of TAT-MT recombinant fusion protein from the freshwater crab Sinopotamon honanenseDNA sequencing confirmed that there were no nucleotide substitutions and no mutations in the MT and TAT-MT gene fragments amplified by long primer PCR technology.The experiments showed that the recombinant plasmids p ET28a-SUMO-TAT-MT,p EXsec1-TAT-MT,and p ET28a-TAT-MT were successfully constructed.3.Optimized expression and purification of TAT-MT from the freshwater crab(Sinopotamon honanense)After screening for the optimal induction conditions of the protein,the results showed that the optimal induction condition for the fusion protein TAT-MT was 37?,1.0 m M IPTG and after 8 hours of induction.TAT-MT was obtained with a molecular mass of approximately 20 k Da.The results of SDS-PAGE test proved that the protein purification effect was good,most of the miscellaneous proteins had been removed.4.Cell membrane penetration and functional activities detection of TAT-MT from freshwater crab(Sinopotamon henanense)Immunofluorescence was used to experiment that MT protein cannot enter the inside of the cell,and TAT-MT protein can enter the cell membrane or on the membrane.Hydrogen peroxide was used to cause cell damage.MTT was used to determine the repairing effect of the fusion protein on cell damage.TAT-MT has good hydroxyl radical scavenging rate and total antioxidant capacity determined in vitro.Therefore,the metallothionein from Sinopotamon henanense can enter the cell smoothly,and it has a repairing effect on the oxidative damage of the cell,and has a certain antioxidant capacity and a hydroxyl radical scavenging rate.Conclutions:In this study,TAT-MT gene was successfully cloned by long primer PCR,There combinant plasmids p ET28a-SUMO-TAT-MT ? p ET28a-TAT-MT and p Ex Sec I-TAT-MT were successfully constructed.And TAT was successfully expressed in E.coli.Immunofluorescence were used to detect the transmembrane effect of TAT and to verify that the protein has a certain repair effect on cell oxidative damage.TAT-MT was tested for its ability to scavenge hydroxyl radicals and total antioxidant capacity in vitro.This study can lay the foundation for the futher application of metallothionein...