Expression and Renaturation of Recombinant Human Lysozyme in Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2019-0680

Abstract

Abstract: Human lysozyme（HLZ）is a glycosidolytic enzyme with antibacterial and anti-inflammatory effects. As an alternative to antibiotics,HLZ has been widely used in the food industry,animal husbandry and medical fields. How to obtain HLZ with high yield,high activity and purity is an technical issue to be urgently solved. The codons of HLZ gene were optimized to improve its fitness and expression in Escherichia coli,then the optimized gene of HLZ was introduced into expressing plasmid pET21a and expressed in E. coli expressing strain BL21（DE3）. After inclusion body was dissolved by 8 mol/L urea solution,the effects of 3 refolding methods including one-step dialysis,gradient dialysis and gradient dilution and the concentration of glutathione REDOX（GSSG/GSH）,arginine and glycerol on the refolding of HLZ were investigated to confirm the best refolding plan. Results showed that under the induction by 0.5 mmol/L IPTG at temperature of 37℃,HLZ with the molecular weight of about 14.7 kD was successfully expressed in E. coli,and the expression quantity of inclusion body was about 380 mg/L（wet weight）. After refolding the denatured HLZ by one step dialysis,gradient dialysis and gradient dilution,the specific activities of the enzyme were measured as 147 U/mg,335 U/mg and 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 HLZ were further investigated. The results showed that the highest enzyme specific activities of refolded HLZ was 1 170 U/mg,which was much higher than the 335 U/mg specific activity of HLZ when the 3 contents were not added,but lower than the enzyme specific activity 1 732 U/mg by commercial HLZ. In conclusion,target HLZ was successfully expressed in E. coli,and recombinant HLZ with high activity was successfully obtained by the refolding system of inclusion body.

Key words: human lysozyme, gene clone, expression system of Escherichia coli, inclusion body, renaturation of inclusion body, Micrococcus lysoleikticus

ZHANG Chun-chen, HU Shuang-yan, RUAN Hai-hua. Expression and Renaturation of Recombinant Human Lysozyme in Escherichia coli[J]. Biotechnology Bulletin, 2020, 36(3): 153-161.

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