Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Chemistry I Division, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Appl Environ Microbiol. 2018 May 31;84(12). doi: 10.1128/AEM.00270-18. Print 2018 Jun 15.
In , many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in The bacterium is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of , we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.
在大肠杆菌中，许多重组蛋白在周质中产生。为了将这些蛋白定向到这个隔室，它们配备了一个 N 端信号序列，以便通过蛋白质导肽 Sec 转运体穿过细胞质膜。最近，我们使用单链可变抗体片段 BL1 表明，协调目标基因表达强度与 Sec 转运体能力可以用来提高周质中重组蛋白的产量。在这里，我们使用蛋白质组学方法研究了通过提高 BL1 在周质中的产量来改善目标蛋白产量的结果。当目标基因表达强度与 Sec 转运体能力不匹配时，分泌蛋白的易位受损、细胞质中蛋白质错误折叠/聚集以及能量代谢效率低下会导致生长不良和蛋白质产量低。目标基因表达强度与 Sec 转运体能力的协调导致正常生长、增强的蛋白质产量，以及令人惊讶的是，除了产生的目标蛋白之外，蛋白质组的组成与空表达载体的细胞相同。因此，单链可变抗体片段 BL1 可以在不对生产宿主造成任何明显不利影响的情况下在周质中高效生产。最后，我们表明，在优化条件下，一小部分目标蛋白通过外膜囊泡释放到细胞外环境中。我们设想，我们的观察结果可以用于设计策略，进一步提高大肠杆菌中分泌型重组蛋白的产量。大肠杆菌被广泛用于生产重组蛋白。通常，使用基于反复试验的筛选方法来确定导致高重组蛋白产量的条件。在这里，对于 BL1 在大肠杆菌周质中的生产，我们表明其生产的优化伴随着应激的缓解。这表明可以使用监测应激反应来促进增强的重组蛋白产量。
