(623aj) Assaying Secreted Titer of Heterologous Proteins In Salmonella | AIChE

(623aj) Assaying Secreted Titer of Heterologous Proteins In Salmonella

Authors 

Tullman-Ercek, D. - Presenter, University of California, Berkeley


Inefficient bacterial secretion causes most heterologous protein to be expressed as inclusion bodies. Additionally, insoluble aggregation and host toxicity limit titer. We expect to achieve high titer of soluble heterologous proteins in bacterial culture by engineering the type III secretion system (T3SS) of Salmonella typhimurium for efficient peptide secretion to the extracellular fluid. The T3SS is a transmembrane pump found in many Gram-negative pathogenic bacteria. It translocates peptides from the cytoplasm across both the inner and outer membranes of the bacterium and the cell membrane of the host cell. Spider silk monomers have been successfully secreted into the extracellular fluid in the absence of host cells using the wild-type T3SS. However, reported titers are several orders of magnitude below those achieved through routine industrial bacterial fermentation and secretion efficiency is less than 20%. We report the development of a high-throughput fluorescent assay for secretion efficiency quantification using flow cytometry. Single cell analysis is achieved through the expression of a secretion-tagged transcription factor that represses gfp production. Such a screening method will enable the rapid, high-throughput quantification of secretion efficiency and assist in the development of a T3SS-based prokaryotic protein expression system.