Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids
Biotechnology Journal, Vol. 11, 2016
Sang-Woo Bae, Doyoung Eom, Ngoc Lan Mai and Yoon-Mo Koo
The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca2+ and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over three-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids EMIM-Cl to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffers at relatively high initial protein concentration (5 mg/mL). These results indicated that refolding methods utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloproteins.