Application Of Immobilized Cholesterol Column Chromatography In Protein Refolding

Utilization of chromatographic columns for protein refolding is a good strategy. Size-exclusion chromatography, ion exchange chromatography and affinity chromatography have successfully refolded many proteins. In principle, commercially available hydrophobic interaction chromatography (HIC) media can be used to assist protein refolding because the hydrophobic interaction between HIC ligands and denatured proteins as well as refolding intermediates likely minimize aggregate.However, commercially available HIC-assisted refolding may bring about the problem that hydrophobic ligands of HIC media of relative high density, in view of the commercial purpose to improve capacity for separation, possibly lead to formation of incorrect structure. So it's necessary to develop a kind of media of proper hydrophobicity, which is favorable for hydrophobic collapse as well as aggregate minimum, to facilitate the refolding process.In this paper,cholesterol modified Sepharose matrix was prepared by two-step method with cyanuric chloride as a linker. Refolding by dilution, passing-through way and adsorption-elution way with immobilized cholesterol column were studied for the reactivation of denatured-reduced lysozyme. The results showed that When loading mass was 0.25 mg Lys/ml support, protein recovery 96.3% and activity yield 91.4% were obtained by adsorption-elution way,which were really higher than the other two methods. Besides,the structure of the refolded lysozyme was found the same as the native lysozyme by Circular Dichroism (CD), Fluorescence and HPLC.Further the effects of flow rate, elution way, GdnHCl gradient volume, initial protein concentration and loading mass on refolding yield were investigated. The results showed that even loading mass reached 1 mg/ml support, activity yield 85% were obtained with 0.02 ml/min elution rate and 0.2 column volume (CV) GdnHCl gradient. Denatured-reduced lysozyme was effectively refolded into the active form, which suggests that adsorption–elution way is a very effective method for protein refolding.