Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu FI-90014, Finland.
J Microbiol Biotechnol. 2024 May 28;34(5):1126-1134. doi: 10.4014/jmb.2311.11025. Epub 2024 Mar 29.
The production of disulfide bond-containing recombinant proteins in has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of : i) engineered strains with deletions in the disulfide reduction pathways, SHuffle, and ii) the co-expression of oxidative folding catalysts, CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.
在大肠杆菌中生产含有二硫键的重组蛋白传统上可以通过包涵体复性或靶向蛋白到周质空间来实现。然而，这两种方法都有局限性。为了允许在细胞质中生产具有二硫键的蛋白质，开发了两种广泛的策略：i）在二硫键还原途径中缺失的工程菌株，如 SHuffle，和 ii）氧化折叠催化剂的共表达，如 CyDisCo。然而，据我们所知，这些策略的相对有效性尚未得到适当评估。在这里，我们系统地比较了在这两个系统中表达时天然状态下含有二硫键的 14 种不同感兴趣蛋白质（POI）的纯化产量。我们还比较了不同背景菌株、常用启动子和两种培养基类型（定义和丰富的自动诱导）的影响：在丰富的自动诱导培养基中，POI 可以在没有二硫键形成系统的情况下以可溶性（非天然）状态产生，而 SHuffle 产生的 POI 的纯化产量更高，而使用 CyDisCo 则产生其他所有蛋白质的纯化产量更高。在化学定义的培养基中，使用 CyDisCo 产生的纯化产量在所有情况下至少高 10 倍。此外，当使用 CyDisCo 生产时，测试的三种 POI 的质量更高。
