Universal Genetic Assay for Engineering Extracellular
Protein Expression
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Abstract
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<i>.</i> 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 <i>in
situ</i> 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