Engineering Ag43 Signal Peptides with Bacterial Display and Selection

Abstract

Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, 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 just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.

Unveiling the impact of cryptic plasmids curing on Escherichia coli Nissle 1917: massive increase in Ag43c expression

“…Ag43c, like other variants of Ag43, possesses a modular structure (Fig. 4 a), including: the N-terminal signal sequence (SP), the L-type passenger domain (α domain), the autochaperone domain (AC), the C-terminal translocator (β domain) (Ageorges et al 2023 ; Benz and Schmidt 2011 ; Koh et al 2022 ). Upon crossing the inner membrane, the SP sequence is cleaved (Ageorges et al 2019 ; Koh et al 2022 ).…”

“…4 a), including: the N-terminal signal sequence (SP), the L-type passenger domain (α domain), the autochaperone domain (AC), the C-terminal translocator (β domain) (Ageorges et al 2023 ; Benz and Schmidt 2011 ; Koh et al 2022 ). Upon crossing the inner membrane, the SP sequence is cleaved (Ageorges et al 2019 ; Koh et al 2022 ). After traversing the outer membrane, the region between the AC and β domain is cleaved by an unknown protease (Ageorges et al 2023 ).…”