Engineering Ag43 Signal Peptides with Bacterial Display and Selection

Koh, Darius Wen-Shuo, Tay, Jian-Hua, and Gan, Samuel Ken-En (2023) Engineering Ag43 Signal Peptides with Bacterial Display and Selection. Methods and Protocols, 6 (1). 1.

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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.

Item ID: 78604
Item Type: Article (Research - C1)
ISSN: 2409-9279
Keywords: Ag43 autotransporter, engineering signal peptides, error-prone PCR, protein surface display, single-cell screening
Copyright Information: © 2022 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Date Deposited: 22 Nov 2023 04:43
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3204 Immunology > 320402 Applied immunology (incl. antibody engineering, xenotransplantation and t-cell therapies) @ 100%
SEO Codes: 20 HEALTH > 2001 Clinical health > 200105 Treatment of human diseases and conditions @ 100%
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