Cloning,Expression,Purification And Characterization Of A Thermophilic Galactose Kinase Tth0825 From Thermus Thermophilus

Background and purpose:Galactokinase is an phosphotransferase that facilitates the phosphorylation of?-D-galactose to galactose 1-phosphate at the expense of one molecule of ATP.Galactokinase was found in most organisms for the catabolism of?-D-galactose to glucose 1-phosphate.First isolated from mammalian liver,galactokinase has been studied extensively in yeast,archaea and plants.Experimental method:The tth0825 gene was amplified by PCR using the Thermus thermophilus?JCM10941?genome as template.The recombinant plasmid pET28a-tth0825 was constructed using pET28a as a vector,and the recombinant protein was expressed by E.coli Transetta?DE3?.The purified protein was obtained by Ni²⁺column affinity chromatography,and the purified protein was subjected to characterization.Results:The gene sequence was successfully obtained by PCR,and the recombinant plasmid pET28a-tth0825 was successfully constructed.The expression plasmid Transetta-28a-tth0825 successfully expressed the protease with a size of about 39.5 kDa.And,the optimum temperature of galactose kinase Tth0825 is 70°C,the optimum pH is 9.5,the best metal ion is Mg²⁺,and the galactose kinase Tth0825catalyzes the D-galactose reaction to reach about 60%conversion rate.Conclusion:The recombinant plasmid pET28a-tth0825 was successfully constructed and expressed in E.coli expression system.After purification by Ni²⁺column affinity chromatography,pure protein was obtained.The galactose kinase showed good thermal stability and catalytic activity.