Wong Ru Shen, Alias Nurul Nadia Mohamad, Ong Eugene Boon Beng, Liew Mervyn W O
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia.
Methods Mol Biol. 2023;2617:189-200. doi: 10.1007/978-1-0716-2930-7_13.
Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5 M guanidine hydrochloride, GdnHCl) with a high temperature (95 °C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.
包涵体（IB）是大多为错误折叠的多肽形成的致密不溶性聚集体，通常由重组蛋白的过表达产生。在诸如大肠杆菌、酵母和高等真核生物等蛋白质表达系统中已观察到包涵体的形成。为了以天然状态回收可溶性重组蛋白，通常首先用高浓度变性剂溶解包涵体。随后进行变性剂的同时去除或降低，并转变为有利于重折叠的化学环境，以促进溶解的蛋白重折叠为其天然状态。由于使用的变性剂浓度高，传统的重折叠方法可能会导致产物稀释且缓冲液效率低下。为了克服传统重折叠方法的局限性，提出了一种基于温度的重折叠方法，该方法在溶解过程中将低浓度变性剂（0.5 M盐酸胍，GdnHCl）与高温（95°C）相结合。在本章中，我们描述了一种基于温度的重折叠方法，用于从包涵体中回收核心链霉亲和素（cSAV）。通过基于温度的方法，通过消除稀释方法所需的浓缩步骤以及减少稀释或去除变性剂所需的缓冲液体积实现了强化。溶解过程中的高温处理也可能导致了不需要的宿主细胞蛋白的变性和聚集，然后可以通过离心步骤将其去除，从而得到高纯度的重折叠cSAV，而无需柱纯化。通过生物素结合测定和SDS-PAGE对重折叠的cSAV进行了表征，同时通过RP-HPLC测定了纯度。
