Bacteria Modified to Secrete Biologically Active Protein for Large-Scale Production

E. coli is the most common prokaryote used to produce protein. The expressed protein generally accumulates in the cytoplasm. While this approach is useful for some proteins not all proteins can be accumulated in the cytoplasm in an active state. When the desired protein is produced at high levels it may be toxic to the host cell or accumulate as an insoluble particle known as an inclusion body making it difficult to recover in an active form. One option is to engineer protein so it is secreted from the cell but E. coli and other Gram-negative bacteria generally are considered poor hosts for secreted protein production. No well-understood secretory pathways in E. coli to transport heterologous proteins to the extracellular environment currently exist. The recent discovery of YebF-mediated secretion is the first report of a native E. coli system capable of secreting both the native protein YebF and translational fusions to YebF. However the expression level of YebF fusion proteins typically is low. UW–Madison researchers have discovered E. coli mutations that substantially increase the amount of biologically active recombinant protein secreted from cells. The mutations disrupt genes in a YebF-mediated protein secretion pathway. Bacteria modified to contain these mutations are useful for the production of secreted proteins. They can be used to produce proteins that might otherwise not be expressed due to toxicity or folding errors. They also can be used to produce secreted complexes of enzymes such as cellulases and xylanases for the manufacture of cellulosic biofuels. Applications: 1) Manufacturing proteins for bioenergy production therapeutic biologics and research tools 2) Rapid high throughput production of proteins on a commercial scale


1) Enables the production of proteins that might not otherwise be expressed due to toxicity or folding errors 2) Substantially increases the amount of biologically active heterologous protein that can be secreted from E. coli and other bacteria 3) Suitable for Shigella Yersinia Salmonella and Escherichia sp. bacteria 4) May be readily employed in automated systems 5) Mutations do not affect bacterial growth. 6) Because proteins are exported the levels of contamination endotoxin host cell proteins and nucleic acids are significantly lower making purification easier and thus lowering production cost and time.

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