Manchester Institute of Biotechnology, University of Manchester, M1 7DN, Manchester, UK.
Absynth Biologics Ltd., BioHub, Alderley Park, Cheshire, SK10 4TG, UK.
Appl Microbiol Biotechnol. 2021 Jul;105(13):5657-5674. doi: 10.1007/s00253-021-11405-9. Epub 2021 Jun 28.
Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of 'difficult-to-express' bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.'s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1-15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.
细菌表达系统仍然是用于重组蛋白生产的广泛使用的宿主。然而，在大肠杆菌等细菌系统中过量表达重组靶蛋白会导致蛋白质溶解性差，并形成不溶性聚集体。因此，已经采用了许多策略或替代工程方法来增加重组蛋白的产量。在本案例研究中，我们介绍了用于增加 Absynth Biologics Ltd. 的艰难梭菌疫苗计划中“难表达”细菌抗原 Ant2 和 Ant3 的重组生产和可溶性的策略。当在细菌细胞中过表达时，单个重组抗原（Ant2 和 Ant3）和融合蛋白（Ant2-3 和 Ant3-2）形成不溶性聚集体（包涵体）。此外，观察到 Ant2-3 的蛋白水解切割。优化培养条件和改变构建设计以包括 N 端可溶性标签并没有提高抗原的可溶性。然而，不同缓冲液/添加剂的筛选表明，仅添加 1-15 mM 的二硫苏糖醇就可以减少不溶性聚集体的形成并提高 Ant2 和 Ant3 的稳定性。为 Ant2 和 Ant3 生成了结构模型，并使用基于可溶性的预测工具来确定疏水性和电荷对蛋白质生产的作用。结果表明，Ant2 结构的表面检测到一个大的非极性区域（包含疏水性氨基酸），而 Ant3 则检测到带正电荷的区域（包含赖氨酸和精氨酸氨基酸），这两者都与蛋白质的可溶性差有关。我们提出了一系列策略和预测方法的指南，旨在指导构建设计，以便在表达研究之前定义和设计序列/结构，从而可以提高细菌表达系统中单个和潜在多结构域（或融合）抗原的表达和稳定性。
