Production of heterologous proteins in a microbial host can be advantageous due to their genetic tractability, fast growth, and robustness in large cultures. However, these hosts are limited because they retain the protein product intracellularly, requiring a complicated and expensive downstream purification scheme. A promising solution is to engineer the bacteria to secrete the protein product to the extracellular space, which would greatly ease downstream purification requirements. Many research groups have focused on secreting heterologous protein products through a variety of secretion pathways native to bacteria, but the titer of secreted protein is often too low for commercial interests. While many microbial engineering techniques could be used to improve secretion titer, studies are often limited by a lack of fast, quantitative, and high throughput assays for measuring secreted protein. For this purpose, we have utilized the mini fluorescence activating protein (mFAP), a small, de novo protein consisting of a single beta barrel, which reversibly binds to the substrate DFHBI to produce a fluorescent signal. In this talk, we show that this protein is readily secreted through multiple secretory pathways when fused to the relevant secretion tag, and it produces a measurable fluorescent signal in media when its substrate is added exogenously. A high-throughput plate based fluorescence assay for measuring secreted protein using this reporter can develop a full signal in minutes, can measure secreted protein amounts across three orders of magnitude, and can be used in undefined media which typically cause low signal to noise ratios. Using the Type 3 Secretion System of Salmonella typhimurium as an example, we show that this assay can accurately measure how engineering changes affect secretion titer, and we show how this assay can enable new studies which enable further improvements to secretion titer.

Development of a high-throughput assay for measuring secreted protein titer based on a de novo fluorescent reporter

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.