Antigen-43-mediated surface display revealed in Escherichia coli by different fusion sites and proteins

Abstract

Background: Cell surface display system allows for endowing functional proteins expressed on bacterial surface by fusing different anchor proteins. Among PgsA, Blc, and Omp anchor, the antigen 43 (Ag43)-mediated surface display is a novel system in Escherichia coli. Here, we have demonstrated the red fluorescent protein (RFP) and cellulase (EC 3.2.1.4) on the cell surfaces at two different fusion sites in Ag43. Results: We introduced two fusion sites which are unstructured domain (52-138 aa) and autochaperone domain (600-700 aa) at N-terminal for passenger proteins. As a result, the surface-displayed RFP expressed in plasmid pET28a, but the intracellular RFP expressed more than the surface-displayed RFP. Improved display efficiency of Ag43 was present when fusing at the site of the 138th amino acid (aa) compared to fusing at the site of the 700th aa. For endoglucanase, whole-cell surface-displayed Ag43-138-BsCel5 showed the highest specific activity which was 4.65-fold of BsCel5. Cell-displayed cellulase preserved residual activity ranging from 78% to 38% at temperatures from 55 °C to 80 °C, respectively. Conclusions: This study is to demonstrate the novel surface display system of Ag43 in E. coli by targeting two different proteins RFP and BsCel5 that were successfully displayed on the cell surfaces at two different fusion sites. The Ag43 system displays surface heterologous proteins and is a potential whole-cell catalyst in the bioconversion of cellulose.

Pneumatically Driven Microfluidic Platform and Fully Automated Particle Concentration System for the Capture and Enrichment of Pathogens

“…Bacterial antigen 43 (Ag43) comprises a signal peptide (1–52 aa), N-terminal unstructured passenger (53–137 aa), linker (138–600 aa), putative auto-chaperone (AC, 600–700 aa), and C-terminal β-barrel pore (701–1039 aa) (Figure S1a). , The β-barrel domain integrates into the bacterial outer membrane, where it forms a pore, and the passenger domain translocates through the β-barrel pore to the cell surface . The plasmid named Ag43β-RBD inserts a passenger protein through genetic replacement of 53–137 aa (SARS-CoV-2 RBD).…”

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

“…Additionally, the Ag43 system has been employed to display β-glucosidase on the outer membrane of E. coli , facilitating the fermentation of crystalline cellulose during simultaneous saccharification and fermentation (SSF) (Muñoz-Gutiérrez et al 2014 ). In another instance, red fluorescent protein (RFP) and cellulase (EC 3.2.1.4) were fused with Ag43, respectively, and can be displayed on the surface of E. coli (Jing et al 2019 ). This implies that the EcNc strain, characterized by high expression of Ag43c, holds the potential to be used as a surface display strain.…”

Engineering Ag43 Signal Peptides with Bacterial Display and Selection

“…After 6 and 12 h of induction, 5 mL of culture was collected at the respective time points. Trypsinization was previously performed and found suitable to characterize autotransporter-based systems in E. coli [ 52 , 53 ]. To determine total fluorescent sfGFP on the surface of cells, the bacteria cells were normalized by OD where they were first spun down and resuspended to the equivalent of OD 4 or OD 8 with 400 μL of 2.5 g/L of Trypsin solution (Nacali Tesque, Japan; Cat: 32777-44, Lot No.…”