Universal Genetic Assay for Engineering Extracellular Protein Expression

作者：Haitjema Charles H; Boock Jason T; Natarajan Aravind; Dominguez Miguel A; Gardner Jeffrey G; Keating David H; Withers Sydnor T; DeLisa Matthew P^*

来源：ACS Synthetic Biology, 2014, 3(2): 74-82.

DOI：10.1021/sb400142b

• bacterial expression
• biarsenical FlAsH labeling
• protein folding and export
• secretion-pathway engineering
• type II and type III protein secretion
• YebF

A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli. However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically Useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli. Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible in situ without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli. Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production.

• 出版日期2014-2