Signal peptide replacement of Ag43 enables an efficient bacterial cell surface display of receptor-binding domain of coronavirus.

To enable an efficient bacterial cell surface display with effective protein expression and cell surface loading ability via autotransporter for potential vaccine development applications, the inner membrane protein translocation efficiency was investigated via a trial-and-error strategy by replacing the original unusual long signal peptide of E. coli Ag43 with 11 different signal peptides. The receptor-binding domain (RBD) of coronavirus was used as a neutral display substrate to optimize the expression conditions, and the results showed that signal peptides from PelB, OmpC, OmpF, and PhoA protein enhance the bacterial cell surface display efficiency of RBD. In addition, the temperature has also a significant effect on the autodisplay efficiency of RBD. Our data provide further technical basis for the biotechnological application of Ag43 as a bacterial surface display carrier system and further potential application in vaccine development.

The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System.

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA877 and SadA269) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158-172aa and UreB349-363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA877 or SadA1292. Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA877 or SadA1292 as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA1292. The oral administrated pSadBA1292-FUM/StmΔygeAΔmurI and pSadBA877-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system.

BACKGROUND: RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. METHODS AND RESULTS: BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp')-OmpA', SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. CONCLUSION: The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.

Develop a PD-1-blockade peptide to reinvigorate T-cell activity and inhibit tumor progress.

Immune checkpoint inhibitors, particularly monoclonal antibodies blocking the programmed cell death 1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway, have been successfully utilized in the clinic. However, certain drawbacks associated with antibodies, such as high immunogenicity and poor tissue penetration, need to be addressed for their broader clinical application. Peptides, as low molecular weight alternatives, have garnered increasing interest in this field. In this study, we employed bacterial surface display technology to identify a PD-1-binding peptide, PBP. The PBP peptide exhibited moderate affinity for human PD-1 (hPD-1) and displayed cross-reactivity with mouse PD-1 (mPD-1). Molecular docking analysis revealed that the interaction residues of the PBP peptide with PD-1 played crucial roles in the formation of the PD-1/PD-L1 complex. A competing binding assay demonstrated that the peptide could interfere the interaction of PD-1 and PD-L1. Moreover, in vitro experiments showed that the PBP peptide could reinvigorate T cells inhibited by PD-L1. In an in vivo mouse model of CT26, the PBP peptide effectively suppressed tumor growth by enhancing T cell function. In conclusion, our results suggest that the PBP peptide exerts an anti-tumor effect by impeding the interplay between PD-1 and PD-L1, highlighting its potential as an alternative for tumor immunotherapy.

Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting.

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived proteins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naïve affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recognition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide technology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity.

Identification of Microorganisms that Bind Specifically to Target Materials of Interest Using a Magnetophoretic Microfluidic Platform.

Discovery of microorganisms and their relevant surface peptides that specifically bind to target materials of interest can be achieved through iterative biopanning-based screening of cellular libraries having high diversity. Recently, microfluidics-based biopanning methods have been developed and exploited to overcome the limitations of conventional methods where controlling the shear stress applied to remove cells that do not bind or only weakly bind to target surfaces is difficult and the overall experimental procedure is labor-intensive. Despite the advantages of such microfluidic methods and successful demonstration of their utility, these methods still require several rounds of iterative biopanning. In this work, a magnetophoretic microfluidic biopanning platform was developed to isolate microorganisms that bind to target materials of interest, which is gold in this case. To achieve this, gold-coated magnetic nanobeads, which only attached to microorganisms that exhibit high affinity to gold, were used. The platform was first utilized to screen a bacterial peptide display library, where only the cells with surface peptides that specifically bind to gold could be isolated by the high-gradient magnetic field generated within the microchannel, resulting in enrichment and isolation of many isolates with high affinity and high specificity toward gold even after only a single round of separation. The amino acid profile of the resulting isolates was analyzed to provide a better understanding of the distinctive attributes of peptides that contribute to their specific material-binding capabilities. Next, the microfluidic system was utilized to screen soil microbes, a rich source of extremely diverse microorganisms, successfully isolating many naturally occurring microorganisms that show strong and specific binding to gold. The results show that the developed microfluidic platform is a powerful screening tool for identifying microorganisms that specifically bind to a target material surface of interest, which can greatly accelerate the development of new peptide-driven biological materials and hybrid organic-inorganic materials.

A recombinant Mycobacterium smegmatis-based surface display system for developing the T cell-based COVID-19 vaccine.

The immune escape mutations of SARS-CoV-2 variants emerged frequently, posing a new challenge to weaken the protective efficacy of current vaccines. Thus, the development of novel SARS-CoV-2 vaccines is of great significance for future epidemic prevention and control. We herein reported constructing the attenuated Mycobacterium smegmatis (M. smegmatis) as a bacterial surface display system to carry the spike (S) and nucleocapsid (N) of SARS-CoV-2. To mimic the native localization on the surface of viral particles, the S or N antigen was fused with truncated PE_PGRS33 protein, which is a transportation component onto the cell wall of Mycobacterium tuberculosis (M.tb). The sub-cellular fraction analysis demonstrated that S or N protein was exactly expressed onto the surface (cell wall) of the recombinant M. smegmatis. After the immunization of the M. smegmatis-based COVID-19 vaccine candidate in mice, S or N antigen-specific T cell immune responses were effectively elicited, and the subsets of central memory CD4+ T cells and CD8+ T cells were significantly induced. Further analysis showed that there were some potential cross-reactive CTL epitopes between SARS-CoV-2 and M.smegmatis. Overall, our data provided insights that M. smegmatis-based bacterial surface display system could be a suitable vector for developing T cell-based vaccines against SARS-CoV-2 and other infectious diseases.

A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase.

Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-to-electricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O2 EBFC, herein we prepared a starch-oxidizing bioanode based on displaying a sequential enzyme system of glucoamylase (GA) and glucose dehydrogenase (GDH) on E.coli cell surfaces in a precise way using cohesin-dockerin interactions. The enzyme stoichiometry was optimized, with GA&GDH (3:1)-E.coli exhibiting the highest catalytic reaction rate. The bioanode employed polymerized methylene blue (polyMB) to collect electrons from the oxidation of NADH into NAD+, which jointly oxidized starch together with co-displayed GA and GDH. The bioanode was oxygen-insensitive, which can be combined with a laccase based biocathode, resulting in a membranless starch/O2 EBFC in a non-compartmentalized configuration. The optimal EBFC exhibited an open-circuit voltage (OCV) of 0.74 V, a maximum power density of 30.1 ± 2.8 µW cm-2, and good operational stability.

Engineering IgG1 Fc Domains That Activate the Complement System.

Fc-mediated effector functions are important for the clearance of pathologic cells by therapeutic IgG antibodies through two mechanisms: via the activation of the classical complement pathway and through the binding to Fcγ receptors (FcγRs) which mediate clearance of targeted cells by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by effector cells such as macrophages, NK cells, and other leukocytes subsets. Complement activation results in direct cell killing through the formation of the membrane attack complex (MAC, complement-dependent cytotoxicity or CDC) and in the deposition of complement opsonins on pathogen surfaces. The latter are recognized by complement receptors on effector cells in turn triggering complement-dependent cell cytotoxicity and phagocytosis (CDCC and CDCP, respectively). Little is known about the role of CDCC and CDCP on therapeutic antibody function because on the one hand, IgG isotype antibodies bind to both FcγR and C1q to activate the complement pathway, and on the other, immune cells express complement receptor as well as FcγRs. We engineered IgG1 Fc domains that bind with high affinity to C1q but have very little or no binding to FcγR. To this end, we employed display of IgG in E. coli (which lack protein glycosylation machinery) for the screening of very large libraries (>2 × 109) of randomly mutated human Fc domains to isolate Fc variants that bind to C1q. Herein we introduce and describe the method.

Surface display of HPV16L1 by autotransporter Ag43 / 中华微生物学和免疫学杂志

Objective:To construct a surface display system containing various lengths of the Ag43 passenger domain for an optimal bacterial surface display of foreign protein HPV16L1.Methods:(1) Ag43 gene sequences of different lengths were inserted into pET22b vector to construct four Ag43 surface display vectors (Ag43/138, Ag43/551, Ag43/552 and Ag43/700) using PCR and subcloning strategy. (2) The generation of four HPV16L1-Ag43 fusion constructs was completed by PCR and subcloning methods. (3) HPV16L1-Ag43 fusion proteins were expressed and analyzed by SDS-PAGE. (4) The surface exposure of HPV-16L1 was verified using trypsin digestion.Results:PCR analysis and sequencing results showed that Ag43 surface display vectors and HPV16L1-Ag43 fusions were constructed successfully. SDS-PAGE showed that the expression of HPV16L1-Ag43 fusion proteins could be induced with 0.2 mmol/L IPTG and the protein content was reduced after the cells were treated with trypsin, especially the content of Ag43/700-HPV16L1 that showed a drastic reduction.Conclusions:The Ag43 surface display system was successfully constructed and could be used for a successful display of HPV16L1. This study also showed that Ag43/700 comprising only the α-helix and the β-barrel of Ag43 provided an optimal surface display for HPV16L1.

Label-free flow cytometry-based enzyme inhibitor identification.

Fluorescence-based methods for the identification of enzyme inhibitors are widespread, but usually require protein or ligand labelling. In this study, we present a label-free displacement assay that takes advantage of the intrinsic fluorescence of a tight binding ligand avoiding any labeling. Autodisplay-based accessibility of the target enzyme on the cell surface of Escherichia coli enabled the quantification of fluorescent ligand binding by flow cytometry. Human protein kinase CK2 was used as proof-of-concept enzyme and its ATP competitive inhibitor (E)-1,3-dichloro-6-[(4-methoxyphenylimino)methyl]dibenzo[b,d]furan-2,7-diol (compound 5) was shown to exhibit intrinsic fluorescence (λmax(ex) = 370 nm, λmax(em) = 585 nm). Binding of compound 5 to CK2 displaying cells was quantified via flow cytometry with linearly increasing relative fluorescence up to a concentration of 1.25 µM. The addition of the non-fluorescent CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) competed for compound 5 binding with a half maximal fluorescence reduction at 15.6 µM TBB. This new and simple binding assay provides a valuable tool for the screening of high affinity enzyme inhibitors, overcoming the limitations of fluorescent ligand labelling.

Development of bacterial biosensor for sensitive and selective detection of acetaldehyde.

Acetaldehyde is a human carcinogen and widely existed in alcoholic beverages and polluted air. In this study, a simple, fast, convenient and sensitive acetaldehyde biosensor was developed based on an acetaldehyde dehydrogenase (AldDH) bacteria surface display system. The whole-cell catalyst facilitated the dehydrogenation of acetaldehyde, while coenzyme NAD+ was reduced and the resultant NADH can be detected spectrometrically at 340 nm. The correct location of AldDH on the bacteria surface was confirmed by the subcellular fraction and immunofluorescence analysis. By comparing the fusion protein expression level and whole-cell activity, the proper display system for anchoring AldDH on the cell surface was obtained. The results of kinetics analysis towards both surface-displayed AldDH and intracellular expressed AldDH demonstrated that the mass-transport resistance was dramatically alleviated by cell-surface display strategy. Under optimal conditions, AldDH-surface display strain with the highest whole-cell activity (3.41 ± 0.3 mU/OD600) was applied to spectrophotometry acetaldehyde detection system. An excellent linear relationship between the increases of absorbance at 340 nm and acetaldehyde concentration over the range from 1 µM to 300 µM was reached. The proposed approach offered adequate sensitivity for the detection of acetaldehyde at 0.33 µM. Most importantly, the developed biosensor showed the narrowest substrate specificity towards acetaldehyde, which has been employed for quick determination of acetaldehyde in real samples with good accuracy. The total detection time was within 20 min. The method reported here provided a simple, rapid, and low-cost strategy for the sensitive and selective measurement of acetaldehyde. Therefore, genetically engineered cells may find broad application in biosensors and biocatalysts.

New Bacterial Surface Display System Development and Application Based on Bacillus subtilis YuaB Biofilm Component as an Anchoring Motif.

Bacterial surface display system has been adopted in various biotechnological applications. In the case of Bacillus subtilis, most of the studies have been developed using spore based surface display system utilizing the inherent rigidity of spore against heat, alkali, and shear stress. But, spore harvest, purification and separation need additional cost and labor. To eliminate this procedure and to use the gram-positive nature of B. subtilis, YuaB, which is one of the major B. subtilis biofilm components and locates in the cell wall, based cell surface display system, is developed. P43 promoter driven overexpression of YuaB-His6 tag does not hamper bacterial cell growth and promoted biofilm formation of recombinant strain. Flow cytometry of recombinant strain and its protoplast using FITC-Anti His6 antibody, verified that YuaB locate in plasma membrane and protrude to the outside of cell wall, which means YuaB can be used as very efficient anchoring motif. Using surface expressed YuaB-His6 tag, removal of divalent metal ion, Cu2+ and Ni2+, was tried to test its possibility for the environmental application of developed system.

Functional Display of an Amoebic Chitinase in Escherichia coli Expressing the Catalytic Domain of EhCHT1 on the Bacterial Cell Surface.

Poor solubility is the main drawback of the direct industrial exploitation of chitin, the second most abundant biopolymer after cellulose. Chemical methods are conventional to solubilize chitin from natural sources. Enzymatic hydrolysis of soluble chitinous substrates is a promising approach to obtain value-added by-products, such as N-acetylglucosamine units or low molecular weight chito-oligomers. Protein display on the bacterial membrane remains attractive to produce active enzymes anchored to a biological surface. The Lpp-OmpA system, a gene fusion of the Lpp signal sequence with the OmpA transmembrane region, represents the traditional system for targeting enzymes to the E. coli surface. EhCHT1, the amoebic chitinase, exhibits an efficient endochitinolytic activity and significant biochemical features, such as stability over a wide range of pH values. Using an extended Lpp-OmpA system as a protein carrier, we engineered E. coli to express the catalytic domain of EhCHT1 on the surface and assess the endochitinase activity as a trait. Engineered bacteria showed a consistent hydrolytic rate over a typical substrate, suggesting that the displayed enzyme has operational stability. This study supports the potential of biomembrane-associated biocatalysts as a reliable technology for the hydrolysis of soluble chitinous substrates.

Surface Display Technology for Biosensor Applications: A Review.

Surface display is a recombinant technology that expresses target proteins on cell membranes and can be applied to almost all types of biological entities from viruses to mammalian cells. This technique has been used for various biotechnical and biomedical applications such as drug screening, biocatalysts, library screening, quantitative assays, and biosensors. In this review, the use of surface display technology in biosensor applications is discussed. In detail, phage display, bacterial surface display of Gram-negative and Gram-positive bacteria, and eukaryotic yeast cell surface display systems are presented. The review describes the advantages of surface display systems for biosensor applications and summarizes the applications of surface displays to biosensors.

Characterization of a Histo-Blood Group Antigen-like Substance in Romaine Lettuce That Contributes to Human Norovirus Attachment.

Human noroviruses (HuNoVs) are among the main pathogens causing acute nonbacterial gastroenteritis. Histo-blood group antigens (HBGAs) are widely accepted receptors for HuNoV specific binding. HBGA-like substances in produce are also considered as the critical ligands for capture of HuNoVs. However, the composition of viral ligands from food substrates remains unknown. In this study, an oligosaccharide (H2N2F2) was captured and isolated from romaine lettuce extract by a bacterial surface display system. Using electrospray ionization mass spectrometry and tandem mass spectrometry, it was shown that H2N2F2 was most likely to be a chimera of type A, H, and Lewis a HBGAs. The composition was consistent with our ELISA results using a panel of monoclonal antibodies against HBGAs. Our results revealed a possible interaction mechanism between HuNoVs and romaine lettuce. Better understanding of the interaction of HuNoVs with easily contaminated produce will ultimately aid in the control of and reduction in disease outbreaks.

Rapid Antigen and Antibody-Like Molecule Discovery by Staphylococcal Surface Display.

Ever since the discovery of antibodies, they have been generated by complicated multi-step procedures. Typically, these involve sequencing, cloning, and screening after expression of the antibodies in a suitable organism and format. Here, a staphylococcal nanobody display is described that omits many the abovementioned intermediate steps and allows for simultaneous screening of multiple targets without prior knowledge nor expression of the binders. This paper reports a detailed, general step-by-step protocol to achieve nanobodies of high affinity. Apart from its focus on radioactive and fluorescent targets, it gives options for various other target formats and additional applications for the staphylococcal library; including flow cytometry and immunoprecipitation. This provides a system for antibody engineers that can be easily adopted to their specific needs.

An Effective Platform for Exploring Rotavirus Receptors by Bacterial Surface Display System.

Rotavirus (RV) is a major foodborne pathogen. For RV prevention and control, it is a key to uncover the interaction mechanism between virus and its receptors. However, it is hard to specially purify the viral receptors, including histo-blood group antigens (HBGAs). Previously, the protruding domain protein (P protein) of human norovirus (genotype II.4) was displayed on the surface of Escherichia coli, and it specifically recognized and captured the viral ligands. In order to further verify the feasibility of the system, P protein was replaced by VP8* of RV (G9P[8]) in this study. In the system, VP8* could be correctly released by thrombin treatment with antigenicity retaining, which was confirmed by Western blot and Enzyme-Linked Immunosorbent Assays. Type A HBGAs from porcine gastric mucin (PGM) were recognized and captured by this system. From saliva mixture, the captured viral receptor bound with displayed VP8* was confirmed positive with monoclonal antibody against type A HBGAs. It indicated that the target ligands could be easily separated from the complex matrix. These results demonstrate that the bacterial surface display system will be an effective platform to explore viral receptors/ligands from cell lines or food matrix.

Construction of a bacterial surface display system based on outer membrane protein F.

BACKGROUND: 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. 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. RESULTS: Enhanced 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. CONCLUSIONS: Enhanced 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.