Tandem Oligomeric Expression Of Metallothionein From Freshwater Crab(Sinopotamon Henanense)in Escherichia Coli Enhances Heavy Metal Bioaccumlation And Tolerance

Metallothioneins(MTs)are a family of low molecular weight,cysteine-rich,metal-binding proteins,which play important roles in metal homeostasis and heavy metal detoxification.In our previous study,a novel full length MT c DNA was successfully cloned from the freshwater crab(Sinopotamon henanense).In the present study,tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR(SOE-PCR)and expressed in Escherichia coli.The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins.Furthermore,the metal tolerance and bioaccumulation of E.coli cells expressing oligomeric MTs were determined.The results shown that:1.Construction of tandem expression vector of metallothionein from the freshwater crab Sinopotamon honanenseThere combinant plasmids p ET28a-SUMO-2MT and p ET28a-SUMO-3MT were successfully constructed with high-fidelity.2.Optimized expression and purification of metallothionein from the freshwater crab(Sinopotamon honanense)in Escherichia coliSDS-PAGE analysis showed that the maximal levels of the SUMO-2MT and SUMO-3MT expression were achieved at 8 h after 0.7 m M IPTG induction at 30? and expressed mainly in the soluble form.Compared with the uninduced control,recombinant E.coli harboring p ET28a-SUMO-MT,p ET28a-SUMO-2MT and p ET28a-SUMO-3MTshowed an additional smear and broad band of approximate molecular weight 28 k Da,36 k Da and 42 k Da,respectively,which were consistent with the theoretical values.The SDS-PAGE analysis showed that the protein purification effect was better,and most of the protein was removed.For the heteroprotein,pure apo-MT,apo-2MT,and apo-3MT were obtained.A special metal cluster absorption peak was detected by ultraviolet spectrophotometer at wave lengths of 220 nm,245 nm and 270 nm,respectively,demonstrating the ability of the MTs tandem recombinant to bind to Zn,Cd and Cu.3.Freshwater crab(Sinopotamon henanense)in Escherichia coli enhances heavy metal bioaccumlation and toleranceExpression of SUMO-MT,SUMO-2MT and SUMO-3MT significantly increased the Cu,Cd and Zn concentration in E.coli compared to control cells harboring p ET28a-SUMO.The cells containing p ET28a-SUMO-3MT exhibited the highest Cu,Cd and Zn bioaccumulation at 1.80,1.94 and1.32?mol/g dry cells,respectively.They were 5.8-fold,3.1-fold and 6.7-fold higher than that of the control cells,respectively.When grown in the control medium,all of the E.coli cells could grow well.When grownin a medium containing Cu SO4,Cd Cl2 or Zn SO4,the growth of E.coli cells harboring different recombinant vectors showed distinctly different growth patterns.Growth of E.coli cells harboring p ET28a-SUMO(as control)and p ET28a-SUMO-MT were inhibited;however,E.coli cells harboring p ET28a-SUMO-2MT and p ET28a-SUMO-3MT reduced this growth defect,indicating that E.coli cells expressing dimeric and trimeric MTs increased Cu,Cd and Zn tolerance compared to the cells harboring p ET28a-SUMO and p ET28a-SUMO-MT.E.coli cells expressing SUMO-MT,SUMO-2MT and SUMO-3MT showed a significant tolerance for Zn2+,Cu2+ and Cd2+ compared to the control cells.The metal tolerance in various strains followed the following order: SUMO-3MT ? SUMO-2MT ? SUMO-MT? control.Our results showed that oligomeric MTs expression in E.coli cells leads to an increase in metal tolerance.Therefore,oligomeric MTs have potential as candidates for heavy metal bioremediation.Our research lays the foundation for the development and application of MTs in the biological treatment of heavy metals.Conclutions:In this study,tandem repeats of two and three copies of the crab MT gene were successfully integrated by overlap extension PCR(SOE-PCR)and expressed in E.coli.Furthermore,the soluble expression of the recombinant oligomeric MTs was increased by the adoption of the SUMO fusion expression system.The oligomeric expression of the MT gene not only significantly enhanced metal tolerance but also increased metal ion accumulation in E.coli cells.These results suggest that both the S.henanense MTs and the E.coli cells harboring oligomeric MTs could serve as candidates for bioremediation applications in heavy metal pollution.