Cloning,Expression And Characterization Of Multi-subunit Chaperonin In Methanosarcina Acetivorans

Chaperonin are important biological macromolecules for cells to maintain protein activity,they exist in all cells in the form of homogenous or heteropoly subunit protein polymers.it plays a core role in cellular stress responses such as temperature,acid-base,high salt,oxidation and so on.Methanosarcina acetivorans is a methanogenic thermophilic archaeon isolated from marine sediments,which plays an important role in the global carbon cycle.Its chaperonin has six subunit genes,one of which is type I chaperonin(MA0086),which is similar to GroEL/ES in bacteria;the other five are type II chaperonin(MA4413,MA4386,MA0857,MA0631,MA1682).In this experiment,the genes MA4386,MA0857 and MA0086 were cloned and expressed,and the MA4386 gene was purified and its related properties were determined.The MA4386 gene comes from Archaea.The carboxyl terminal of the Archaeal protein is rich in charged lysine and has a strong positive charge.It can bind to other negatively charged proteins and nucleic acids by electrostatic force with strong affinity.The expression of genes from archaea in Escherichia coli will cause expression problems due to the rarity of codons.When E.coli is used as an expression strain,in the process of induced expression,the codon usage frequency in E.coli is different from that of Archaea codon,so the expression efficiency of the molecular clone in E.coli will be reduced.In order to solve this problem,E.coli BL21(DE3)-RILP strain was selected in this experiment,by optimizing various experimental conditions,such as changing the medium used for induction,reducing the concentration of IPTG induction,increasing the concentration of salt ions in the buffer,and so on.2 × YT medium was used as induction medium,the final concentration of IPTG was 0.1 mM,200 mM NaCl solution was added in the buffer,ATP and nuclease were added during cell fragmentation,the content of soluble expression of MA4386 gene protein in the supernatant was increased,and all the original inclusion bodies were formed.After the optimized conditions,a large number of soluble proteins were expressed in the supernatant.Chaperonin MA4386 protein was purified by protein chromatography purification system AKTA Pure.Finally,the ATP enzyme activity of chaperonin MA4386 protein was determined.The content of phosphate ion released by 10 ?L MA4386 protein was 1.707 nmol,and the enzyme activity(Specific activity)was 205.66 U/mg,which proved that chaperonin MA4386 did has ATP enzyme activity and could perform normal function.