Effect Of Chaperone CPN47 From Thermus Phage TSP4 On Thethermal Stability Of Enzyme Activity

Chaperone (molecular chaperone) is an evolutionarily conserved protein family that can bind non-specific proteins with differences in size, structure and function, and can mediate formation of special structure of proteins. They act in stability nascent proteins, assist of accurate assembly, transport, folding or degradation of proteins in vivo. Chaperone played an important rule in keeping the function of proteins.Chaperone CPN47 and lysin encoding in Thermus pHage TSP4 genome, and dUTPase from host strain Thermus TC16 were cloned and expreessed. Addition to cholinesterase, three kind of enzymes were selected to analyse the effect of CPN47 on their thermostability.Results showed that CPN47 could help to keep the lysins thermal stability under the conditions of treatment at 78℃ for 20 minutes. Without CPN47, the lysin lyase inhibition zone size was only half of the control (Non high-temperature treatment), On the contrary, with CPN47, the lysin lyase inhibition zone size was the same as the control.With CPN47, dUTPase and cholinesterase could maintain high enzyme activity than control (without CPN47) when treated in high temperature, respectively. Further more, the E. coli strains with plasmid enconding CPN47 were encultured at 46℃.Results showed that expresion of CPN47 could improve the strains temperature tolerance and enhanced the survival rate of the strains. As a chaperone from thermopHilic pHage TSP4, after keeping at room temperature for 48 hours, the ATPase activity of CPN47 decreased significantly.Finely, CPN47 with his-tagged was adsorbed to nickel column and then the enzyme was taken through the nickel column. Proteins were eluted with imidazole and the eluate was analyzed by SDS-PAGE electrophoresis. Results showed the protein-complex of CPN47 and the enzyme was formed in this process.In summary, it was found that CPN47 could improve the thermal stability of the enzymes, avoiding the protein heat damage. It might be used in the field of improving the enzyme thermostability.