Metcalf Kevin J, Tullman-Ercek Danielle
Department of Chemical and Biomolecular Engineering, University of California Berkeley, 116 Gilman Hall, Berkeley, CA, 94720, USA.
Methods Mol Biol. 2017;1531:71-79. doi: 10.1007/978-1-4939-6649-3_6.
Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.
异源蛋白可以在细菌宿主中产生，并从细胞成分中纯化出来。将目标蛋白分泌到细胞外空间简化了纯化过程，并被认为可以减轻与目标蛋白在细胞内积累相关的毒性问题。在本方案中，我们描述了一种利用转录控制在细菌培养物中设计蛋白分泌的策略。转录因子HilA被诱导产生以控制分泌机器的产生，进而信号传导目标蛋白的产生和分泌。这使得在优化的培养条件下能够获得高滴度的分泌蛋白，并且在所有测试的蛋白中都观察到了这种效果。
