Optimization of in vitro refolding conditions of recombinant Lepidium draba peroxidase using design of experiments

Abstract

The main objective of this study was to optimize the in vitro refolding conditions of the recombinant Lepidium draba peroxidase (LDP). Initially, the effects of various factors were investigated on LDP refolding yield using one-factor-at-a-time (OFAT) method. Based on the OFAT results, optimum concentrations for LDP refolding were 2 M urea, 2 mM CaCl₂, 0.42 mM l-glutathione oxidized (GSSG), 0.20 mg/ml protein, and 12 μM hemin as well as pH 7. Secondly, according to the OFAT results, design of experiments (DOE) was applied for investigation of the interactions between factors including protein (P), urea (U), CaCl₂ (C), and GSSG (G). The results showed the possible interaction between PC, PG, PU, and GU. Lastly, response surface methodology (RSM) was used for final refolding conditions optimization. The final optimized refolding conditions for LDP were conducted as 2 M urea, 1 mM CaCl₂, 0.70 mM GSSG, 0.07 mM DTT, 0.15 mg/ml protein which they obtained from RSM results and 12 μM hemin, and pH 7 according to the results of OFAT method. Overall, under optimal conditions, 23.4 mg active refolded LDP per liter of expression medium was obtained. So, the refolding yield was calculated to be approximately 48%.

Keywords: Design of experiments; Lepidium draba peroxidase; Refolding; Response surface methodology.