Production Of Recombinant Human Lysozyme In Escherichia Coli

Purpose: Human lysozyme(HLZ)is a glycosidolytic enzyme with antibacterial and anti-inflammatory effects.As an alternative to antibiotics,human lysozyme has been widely used in the food industry,animal husbandry and medical fields.How to obtain human lysozyme with high activity and purity is an urgent scientific problem to be solved.Methods: The codons of human lysozyme gene were optimized to improve its fitness and expression in Escherichia coli(E.coli);Optimized gene of human lysozyme was introduced into expressing plasmid pET21 a and expressed in E.coli expressing strain BL21(DE3);When the target protein is soluble,human lysozyme was isolated and purified by ammonium sulfate fractional precipitation and cation exchange chromatography.When the target protein is insoluble,after inclusion body was dissolved by 8 mol/L urea solution,the effects of three refolding methods of one-step dialysis,gradient dialysis and gradient dilution and the concentration of glutathione REDOX(GSSG/GSH),arginine and glycerol on the refolding of human lysozyme were investigated to confirm the best refolding plan.Results: Under the induction temperature of 18? and 0.5 mmol/L IPTG,human lysozyme of the molecular weight of about 14.7 kDa was successfully expressed in E.coli.The enzyme specific activity values of purified human lysozyme was 1231 U/mg,but lower than the enzyme specific activity values 1732 U/mg of commercial human lysozyme.Under the induction temperature of 37? and 0.5 mmol/L IPTG,insoluble human lysozyme was successfully expressed in E.coli,and the expression quantity of inclusion body was about 380 mg/L(wet weight).After refolding the denatured human lysozyme by one step dialysis,gradient dialysis and gradient dilution,the enzyme specific activity were measured as 147 U/mg?335 U/mg?176 U/mg respectively,indicating that the optimal refolding method was gradient dialysis.The effects of glutathione REDOX(GSSG/GSH ratio),arginine and glycerol concentration on the refolding of human lysozyme were further investigated,and the results showed that when 1:2 GSSG/GSH,4 mmol/L arginine and 6% glycerol were added to the refolded solution,the best enzyme specific activity values of refolded human lysozyme was 1170 U/mg,much higher than the 335 U/mg specific activity value of human lysozyme when the three contents were not added,but lower than the enzyme specific activity values 1732 U/mg of commercial human lysozyme.Conclusion: In this paper,target human lysozyme was successfully expressed in E.coli,and recombinant human lysozyme with high activity was successfully obtained by the refolding system of inclusion body.