Study On Renaturation Of Lysozyme With The Assistance Of Artificial Chaperone

How to refold inclusion bodies correctly with high yield at high protein concentration is a challengeable problem at present for the industrialization of protein. In order to solve this problem, hen egg white Lysozyme (HEWL) had been selected as model protein in this article.Firstly, the effects of several factors such as the concentration of Gdmcl and DTT, denaturation time on free thiol numbers, remaining activity and dilution refolding yields were investigated. The results showed that the denaturation solution including 5 mM DTT, 6M Gdmcl were the optimum environment for the process of lysozyme denaturation, and lysozyme with the concentration of 15mg/mL, which shaked in this solution for 60 min at 36 ℃ at the rate of 100rpm, could be reduced completely. And also the presence of DTT in renaturation solution maybe had no influence on the lysozyme refolding yields.Next, the oxidative refolding of denatured-reduced Lysozyme in presence of cetyltrimerthyl ammonium bromide (CTAB) and β-cyclodextrin (β-CD) were investigated. Various factors affecting the refolding yields were also studied. The refolding system with both CTAB and β-CD(artificial chaperone system) showed more favorable effect on the Lysozyme renaturation. The experiment results showed that the optimal pH were 8.1, cysteine as disulfide-bond reshuffling agent which was between l-2mM, and the optimal concentration of CTAB was 0.6mM, that of β-CD was 30mg/1.5mL. In addition, the refolding yield of Lysozyme decreased with increasing its concentration.Lastly, on the basis of β-CD, the β-CDP beads as stripping detergent agent were synthesized. The beads were transparent and colorless, the size was between 50~250m.β-CDP beads assisted refolding as additive of artificial chaperone got the yield of 40 % at the Lysozyme concentration of 50g/mL because of the bad transfference of the solid-liquid phase. But the beads could be reused for 5 times at least. Above results may become the basis for the immobilization and the application of artificial chaperones.