Studies on the Refolding Process of Recombinant Horseradish Peroxidase

Abstract

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl2 in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.

Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli

“…Recombinant CES1 was significantly expressed in the insoluble fraction, and we purified under denaturing conditions in which 8 M urea was used to solubilize the insoluble protein. Additives such as trehalose, sorbitol, glycerol, arginine and sucrose may enhance the solubility of numerous proteins when included in the refolding buffer [17,19,29,31–33] . In addition, it has been shown that the presence of reducing agents also improves protein solubility [19,29] .…”

“…The refolding of proteins can mainly be achieved in two different ways: dialysis and dilution. During the refolding process, chemical chaperons or additives are usually added to assist in protein conformation, and additives such as trehalose, glycerol, sorbitol, arginine and sucrose have been reported to be advantageous for the refolding step [17–19,29–33] .…”

Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and Rapid Quantification of Uric Acid in Biological Samples

“…All reagents were prepared as mentioned in the latest experiment (3.6), except that the range of UA concentrations was 10-600 µM (10,25,50,100,150,200,250, 300, 350, 400, 500, 550, 600 µM). The reactions were performed using only CV fusion protein and the absorbance was measured at 10 min after the addition of the enzyme.…”

“…Nonetheless, glycosylation of rHRP in yeast cells has been reported to occur non-specifically, resulting in more complicated downstream processes [20]. Besides yeast cells, production of rHRP in E. coli have also been explored [23][24][25][26][27]. The enzyme can be produced up to 1 g/1-Liter culture.…”

Parallelized biocatalytic scanning probe lithography for the additive fabrication of conjugated polymer structures

“…The supernatant, now containing the solubilized inclusion bodies, was then purified by Ni-affinity chromatography under denaturing conditions in Buffer C containing increasing concentrations of imidazole. The purified denatured protein (final concentration approximately 0.18 mg/mL) was then added drop-wise to the refolding buffer (1.7 M urea, 2 mM CaCl2, 7% glycerol, 0.35 mM oxidized glutathione, 0.044 mM dithiothreitol) and stirred at 4°C for 16 h 38. The solution was then filtered (0.2 m pore size) and purified by Ni-affinity chromatography to remove any aggregated protein.…”