Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids

• Published in: Biotechnology journal Vol. 11; no. 4; p. 464
• Main Authors: Bae, Sang-Woo, Eom, Doyoung, Mai, Ngoc Lan, Koo, Yoon-Mo
• Format: Journal Article
• Language: English
• Published: Germany 01.04.2016
• Subjects: Calcium - pharmacology; Hemin - pharmacology; Horseradish Peroxidase - chemistry; Horseradish Peroxidase - metabolism; Ionic Liquids - pharmacology; Kinetics; Metals - chemistry; Protein Conformation; Protein Refolding - drug effects; Solvents - chemistry; Temperature; Urea - pharmacology; Ionic liquids; Refolding additive; Peroxidase; Protein refolding; Metal cofactor
• ISSN: 1860-7314
• DOI: 10.1002/biot.201500142

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 (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-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 buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.