Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens.
Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens.
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DOI：
10.1007/s00253-021-11405-9
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发表时间：
2021-07
影响因子：
5
通讯作者：
Dickson AJ
中科院分区：
文献类型：
--
作者：
Hussain H;McKenzie EA;Robinson AM;Gingles NA;Marston F;Warwicker J;Dickson AJ
细菌表达系统仍然是重组蛋白生产中广泛使用的宿主。然而，重组靶蛋白在大肠杆菌等细菌系统中的过度表达会导致溶解度差和形成不溶性聚集体。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生产。在本案例研究中，我们提出了用于增加“难以表达”的细菌抗原（称为Ant2和Ant3）的重组生产和溶解度的策略，这些抗原来自Absynth Biologics Ltd.的艰难梭菌疫苗计划。单一重组抗原（Ant2和Ant3）和融合蛋白（Ant2-3和Ant3-2）在细菌细胞中过表达时形成不溶性聚集体（包涵体）。进一步观察到Ant2-3的蛋白水解裂解。优化培养条件和改变结构设计以包含n端溶解度标签并没有提高抗原的溶解度。然而，不同缓冲液/添加剂的筛选表明，单独添加1-15 mM的二硫苏糖醇可以减少不溶性团聚体的形成，提高Ant2和Ant3的稳定性。建立了Ant2和Ant3的结构模型，并使用基于溶解度的预测工具来确定疏水性和电荷对蛋白质生成的作用。结果表明，Ant2结构表面存在较大的非极性区（含疏水氨基酸），而Ant3结构表面存在带正电荷区（含赖氨酸和精氨酸氨基酸），两者都与蛋白质溶解度差有关。我们提出了一种策略和预测方法指南，旨在指导构建设计，在表达研究之前，定义和工程序列/结构，这些序列/结构可能导致细菌表达系统中单域和潜在的多域（或融合）抗原的表达和稳定性增加。在线版本包含补充材料，可在10.1007/s00253-021-11405-9获得。
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. The online version contains supplementary material available at 10.1007/s00253-021-11405-9.
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影响因子：
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影响因子：
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