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bioRxiv · 10.1101/2022.07.28.501931

Augmenting the Signal Peptide of the Ag43 Autotransporter for the improved heterologous display of sfGFP using Fluorescence-Activated Cell Sorting (FACs)-assisted natural selection.

Koh, D. W.-S.·Tay, J.-H.·Gan, S. K.
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Abstract

Protein display, secretion and export in prokaryotes are essential for utilizing microbial systems as engineered living materials for medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that increased surface display from 1.4 to 3 folds as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the protein was displayed extracellularly, we trypsinised the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging on epPCR and single-cell sorting to rapidly augment bacterial surface display, a method that could be applied to other bacterial proteins.

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Koh, D. W.-S., Tay, J.-H., Gan, S. K.. 2022-07-28. Augmenting the Signal Peptide of the Ag43 Autotransporter for the improved heterologous display of sfGFP using Fluorescence-Activated Cell Sorting (FACs)-assisted natural selection.. https://doi.org/10.1101/2022.07.28.501931

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