Abstract
BackgroundBacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design. Various outer membrane proteins, such as outer membrane protein A (OmpA), OmpC and outer membrane pore protein E precursor (PhoE), have been used as carriers for surface display, fused to the proteins or peptides of interest in Gram-negative bacteria. Here, we investigated the utility of constitutively expressed OmpF for the display of foreign immune epitopes on the Escherichia coli cell surface and then compared it with plasmid-induced expression of OmpF and OmpC.ResultsEnhanced expression of OmpF was linked to a mutation in the OmpF promoter sequence. This mutation rendered OmpF an ideal carrier protein for the enriched display of a target of interest on the bacterial surface. To this end, we grafted two peptides, harboring important epitopes of the hepatitis B virus (HBV) S antigen and human papilloma virus (HPV) L2 protein, onto OmpF of E. coli by genome editing. The resultant fused OmpF proteins were constitutively expressed in the edited E. coli and purified by membrane component extraction. The epitope that displayed on the bacterial surface was verified by SDS-PAGE, western blotting, flow cytometry, and immunoelectron microscopy of the intact bacteria. We further compared this constitutive expression with plasmid-induced expression of OmpF and OmpC in bacterial cells using the same methods for verification. We found that plasmid-induced expression is much less efficient than constitutive expression of OmpF from the bacterial genome.ConclusionsEnhanced expression of OmpF in a plasmid-independent manner provides an amenable way to display epitopes on the bacterial surface and sheds light on ways to engineer bacteria for biotechnological applications.
Highlights
Bacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design
Point mutation upstream results in constitutive expression of OmpF In the E. coli porin regulon, OmpF and OmpC are regulated by the OmpR–EnvZ system in response to osmolarity: OmpC is preferentially produced in high osmolarity medium, such as LB medium, whereas OmpF is repressed [19]
The protein abundant in ER0808 but not in ER2566 was identified as OmpF. This was confirmed across 4 unique trypsin-digested peptides that exactly matched the theoretical enzymatic fragments of the bacterial OmpF protein (Table 1)
Summary
Bacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design. Microbial cell surface display systems were first described in 1986 by Freudl et al [1] and Charbit et al [2] as a way to expose heterologous proteins or peptides on the surfaces of micro-organisms, including bacteria, viruses, and phages [3] Since this novel technology has been developed and investigated in several applications, including biocatalysis [4, 5], peptide library screening [6, 7], and live bacterial vaccine production [8, 9]. They comprise a common structural motif of a beta-barrel, which contains a variable number of transmembrane anti-parallel beta-strands connected with short periplasmic turns and long external loops [12].
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