Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.
Mol Biotechnol. 2013 Jun;54(2):484-92. doi: 10.1007/s12033-012-9588-6.
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.
辣根过氧化物酶（HRP）是一种重要的含血红素糖酶，已广泛应用于许多生物技术领域。大肠杆菌是一种常用的表达系统，可用于大量获得有价值的蛋白质，如 HRP。然而，重组酶的表达通常会导致包涵体的形成，而植物过氧化物酶等蛋白质的复性产率通常较低。本研究以包涵体的形式克隆并表达了一种重组 HRP。首先，采用单因素法评估了几种添加剂对 HRP 复性的影响。随后，选择具有显著影响的因素（甘油、GSSG/DTT 和酶浓度），通过响应面法（RSM）的中心组合设计进行进一步优化。在获得的最佳条件下，复性效率提高了约两倍。然后在最佳条件（0.35 mM GSSG、0.044 mM DTT、7%甘油、1.7 M 尿素和 2 mM CaCl2，在 20 mM Tris，pH 8.5）下通过测定内源荧光强度监测复性过程，并通过 Soret 吸收检测血红素与复性过氧化物酶的结合情况。此外，通过 Zetasizer 分析在展开和复性条件下的样品，以确定不同介质中的粒径分布。
