Bacterial Surface Display System Research Articles

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.

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

BackgroundRNA-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 resultsBL21 (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.ConclusionThe 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.

Expression of the Hepatitis C Virus core-NS3 Fusion Protein on the Surface of Bacterial Ghosts: Prospects for Vaccine Production.

Antigen presentation using bacterial surface display systems, on one hand, has the benefits of bacterial carriers, including low-cost production and ease of manipulation. On the other hand, the bacteria can help in stimulating the immune system as an adjuvant. For example, using bacterial surface display technology, we developed a hepatitis C virus (HCV) multiple antigens displaying bacteria's surface and then turned it into a bacterial ghost. The HCV core and NS3 proteins' conserved epitopes were cloned into the AIDA gene plasmid as an auto transporter. The recombinant plasmid was then transformed into Escherichia coli (E. coli) Bl21 (DE3). Recombinant bacteria were then turned into a bacterial ghost, an empty cell envelope. Whole-cell ELISA, flow cytometry, and Western blot techniques were used for monitoring the expression of proteins on the surface of bacteria. A fusion protein of HCV core-NS3-AIDA was successfully expressed on the E. coli Bl21 (DE3) surface and confirmed by western blotting, Enzyme-Linked Immunosorbent Assay (ELISA), and flow cytometry detection techniques. The presence of HCV antigens on non-pathogen bacteria surfaces holds promise for developing safe and cost-benefit-accessible vaccines with optimal intrinsic adjuvant effects and exposure of heterologous antigens to the immune system.

Identification of Potential Proteinaceous Ligands of GI.1 Norovirus in Pacific Oyster Tissues.

Human norovirus (HuNoV) is the leading foodborne pathogen causing nonbacterial gastroenteritis worldwide. The oyster is an important vehicle for HuNoV transmission, especially the GI.1 HuNoV. In our previous study, oyster heat shock protein 70 (oHSP 70) was identified as the first proteinaceous ligand of GII.4 HuNoV in Pacific oysters besides the commonly accepted carbohydrate ligands, a histo-blood group antigens (HBGAs)-like substance. However the mismatch of the distribution pattern between discovered ligands and GI.1 HuNoV suggests that other ligands may exist. In our study, proteinaceous ligands for the specific binding of GI.1 HuNoV were mined from oyster tissues using a bacterial cell surface display system. Fifty-five candidate ligands were identified and selected through mass spectrometry identification and bioinformatics analysis. Among them, the oyster tumor necrosis factor (oTNF) and oyster intraflagellar transport protein (oIFT) showed strong binding abilities with the P protein of GI.1 HuNoV. In addition, the highest mRNA level of these two proteins was found in the digestive glands, which is consistent with GI.1 HuNoV distribution. Overall the findings suggested that oTNF and oIFT may play important roles in the bioaccumulation of GI.1 HuNoV.

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

ABSTRACT 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.

Simultaneous biodetection and bioremediation of Cu2+ from industrial wastewater by bacterial cell surface display system

Excess intake (>0.3 μM) of copper may result in a series of neurological diseases. Copper ion (Cu2+) monitoring has attracted much attention due to its toxicity to living organisms. In this study, a Cu2+-binding peptide, rich in histidines was designed and fused with green fluorescent protein (GFP) for the detection and removal of Cu2+. The GFP-peptide fusion (GHG) were displayed on the cell surface of non-pathogenic Escherichia coli BL21 (DE3). The surface-engineered E. coli GHG showed high affinity toward Cu2+ detection and adsorption. The engineered whole cell bacteria aided rapid Cu2+ detection (in 5 min), and its detection limit was 1 μM in the conditions tested. Other metal ions did not interfere with Cu2+ detection. This work has demonstrated highly selective detection (1 μM) and adsorption (302.08 μmol g−1 dry cell) of Cu2+ via surface engineered non-pathogenic E. coli. Approximately 85.6% of Cu2+ was removed from 50-μM aqueous solutions by designed recombinant whole cell bacteria. Engineered bacterial strains can be used continuously via simple sub-culture. This biotechnological approach can potentially be used for the simultaneous detection and removal of Cu2+ ions from the wastewaters.

A temperature-induced chitosanase bacterial cell-surface display system for the efficient production of chitooligosaccharides.

To establish a temperature-induced chitosanase bacterial cell-surface display system to produce chitooligosaccharides (COSs) efficiently for industrial applications. Temperature-inducible chitosanase CSN46A bacterial surface display systems containing one or two copies of ice nucleation protein (InaQ-N) as anchoring motifs were successfully constructed on the basis of Escherichia coli and named as InaQ-N-CSN46A (1 copy) and 2InaQ-N-CSN46A (2 copies). The specific enzyme activity of 2InaQ-N-CSN46A reached 761.34 ± 0.78 U/g cell dry weight, which was 45.6% higher than that of InaQ-N-CSN46A. However, few proteins were detected in the 2InaQ-N-CSN46A hydrolysis system. Therefore, 2InaQ-N-CSN46A had higher hydrolysis efficiency and stability than InaQ-N-CSN46A. Gel permeation chromatography revealed that under the optimum enzymatic hydrolysis temperature, the final products were mainly chitobiose and chitotriose. Chitopentaose accumulated (77.62%) when the hydrolysis temperature reached 60 °C. FTIR and NMR analysis demonstrated that the structures of the two hydrolysis products were consistent with those of COSs. In this study, chitosanase was expressed on the surfaces of E. coli by increasing the induction temperature, and chitosan was hydrolysed directly without enzyme purification steps. This study provides a novel strategy for industrial COS production.

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.

Novel Bacterial Surface Display System Based on the Escherichia coli Protein MipA.

Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V140, V176, K179, V226, V232, and K234, which were truncated from the C-terminal end of the mipA gene (MV140, MV176, MV179, MV226, MV232, and MV234) were examined. The whole-cell lipase activities showed that MV140 was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV140 as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV140. Additionally the MV140 motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV140 motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.

Combinatorial cell surface display system in Escherichia coli for noninvasive colorectal cancer detection

Abstract Navigation and recognition of disease lesions remain challenging during colorectal cancer diagnosis and treatment, given that the precision and capacity of using specific surface antigens as recognition sites are relatively lacking. Moreover, the low penetration rate of noninvasive detection methods has delayed disease diagnosis in developing countries. We developed a bacterial device targeting the Thomsen–Friedenreich antigen, which is abundant on lesion tissue, and produced vesicles that serve as markers for ultrasonic detection. The device uses a bacterial cell surface display system and acoustic reporter gene to function as a screening device for colorectal cancer detection. The diagnostic efficiency of this device was determined by flow cytometry, immunohistochemistry, microfluidic chip-based assay, and ultrasonic examinations on both cellular and tissue scales. In all scales and experiments, our device showed great feasibility upon differentiating disease lesion and normal tissue, while foundation of the usage of vesicle reporter system as noninvasive method has also been laid. The application of this device provides insight into the practicability and prospect of bacterial detecting agents in the field of diagnostics. All animal studies were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University, China (approval No. 201801015) on February 23, 2018.

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.

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.

Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif.

An efficient bacterial surface display system based on the anchoring motif derived from Escherichia coli (E. coli) outer membrane protease OmpT was developed in this study. Referring to the classical Lpp-OmpA (LOA) display system, the signal peptide and nine amino acids of mature Lpp were fused to the transmembrane domain comprising five β-strands of truncated OmpT to generate a novel Lpp-OmpT (LOT) display system. The C-terminal fusion strategy was used to fuse a small peptide (His tag) and red fluorescent protein (mCherry) to the C-terminus of LOT. Cell surface exposure of His tag and mCherry were compared between the LOA and LOT display systems. E. coli expressing LOT-His tag adsorbed more Cu2+ than E. coli expressing LOA-His tag. E. coli expressing both LOT-mCherry-His tag and LOA-mCherry-His tag adhered to Cu2+ chelating sepharose beads, and adhered cells could be dissociated from the beads after excess Cu2+ treatment. More importantly, compared with the LOA system, a higher amount of LOT-mCherry-His tag hybrid protein was demonstrated to be localized at the outer membrane by both fluorescence spectrophotometric determination of cell fractions and cell-surface immunofluorescence assay. These results suggest that genetically modified OmpT can be used as a potential anchoring motif to efficiently and stably display polypeptides and proteins, and that the LOT system could be used in a variety of biotechnological and industrial processes.

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

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.

A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors.

Human noroviruses (HuNoVs) are the dominant cause of food-borne outbreaks of acute gastroenteritis. However, fundamental researches on HuNoVs, such as identification of viral receptors have been limited by the currently immature system to culture HuNoVs and the lack of efficient small animal models. Previously, we demonstrated that the recombinant protruding domain (P domain) of HuNoVs capsid proteins were successfully anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with a plasmid expressing HuNoVs P protein fused with bacterial transmembrane anchor protein. The cell-surface-displayed P proteins could specifically recognize and bind to histo-blood group antigens (HBGAs, receptors of HuNoVs). In this study, an upgraded bacterial surface displayed system was developed as a new platform to discover candidate receptors of HuNoVs. A thrombin-susceptible “linker” sequence was added between the sequences of bacterial transmembrane anchor protein and P domain of HuNoV (GII.4) capsid protein in a plasmid that displays the functional P proteins on the surface of bacteria. In this new system, the surface-displayed HuNoV P proteins could be released by thrombin treatment. The released P proteins self-assembled into small particles, which were visualized by electron microscopy. The bacteria with the surface-displayed P proteins were incubated with pig stomach mucin which contained HBGAs. The bacteria-HuNoV P proteins-HBGAs complex could be collected by low speed centrifugation. The HuNoV P proteins-HBGAs complex was then separated from the recombinant bacterial surface by thrombin treatment. The released viral receptor was confirmed by using the monoclonal antibody against type A HBGA. It demonstrated that the new system was able to capture and easily isolate receptors of HuNoVs. This new strategy provides an alternative, easier approach for isolating unknown receptors/ligands of HuNoVs from different samples including mammalian cell lines, oysters, and fresh produce.

An Engineered Bacterial Display System for the Development of a High‐Throughput Screen for Compounds that Block Amyloid Fiber Formation

The molecular basis for a number of neuronal diseases, such as Alzheimer's and Parkinson's, is aberrant peptide or protein aggregation that result in the formation of amyloid fibers. For example, the Aβ-42 peptide is a molecular component of the plaques found in the brains of Alzheimer's patients, and the protein α-synuclein is a constituent of Lewy bodies, a hallmark of Parkinson's disease. The exact role these proteins play in disease is currently not completely clear but it is generally believed that aberrant self-association contributes in large part to the disease state. For Aβ-42 it is not currently known whether it is low-order complexes (e.g., trimers, tetramers, etc.) or higher-order aggregates that lead to cell death and neuronal degradation. With Parkinson's disease, genetic variations in the gene for α-synuclein give rise to overexpression or aggregate prone variants of the protein. Regardless of the exact disease mechanism, or what level of self-association causes disease, it is generally recognized that blocking the aberrant self-association of these protein-based elements is of high scientific merit. To this end we have engineered a fluorescence-based Bacterial Surface Display (BSD) system that enables a relatively high expression level of a protein-of-interest (POI) on the outer surface of E. coli. Upon expression the POI is fused to an N-terminal periplasmic leader sequence, and to its C-terminus the red fluorescent protein mCherry, and finally a transmembrane protein that functions to anchor the entire fusion construct in the bacterial outer membrane. When the BSD system is used to display Aβ-42, or α-synuclein, the bacterial cells are driven to strongly self-associate. Thus far we have successfully demonstrated the utility of the system using antibodies against Aβ-42. Drug-like compounds that have been proven to block amyloid fiber formation in vitro will also be tested using the Aβ-42 and α-synuclein BSD systems. Immediate and longer-range goals entail the development of a high-throughput screen for additional compounds that bind Aβ-42 and α-synuclein and block their ability to self-associate into amyloid fibers. In addition, the BSD system will also be used for the directed evolution of a small, stable test protein that will be engineered to bind immobilized Aβ-42 and α-synuclein. The test protein, the β1 domain of streptococcal protein-G (Gβ1), consists of an α-helix overlaying a β-sheet made up of two β-hairpins. Codons for select residue positions, located at the edge of one side of the β-sheet, will be randomized and the resulting library displayed using the BSD system. The bacteria displaying the library will be incubated against immobilized Aβ-42 and α-synuclein where successful complex formation (binding) will function as the selection process. The β-hairpin, which contains the amino acids that drive binding, will be expressed/produced separately and assessed for its ability to bind the Aβ-42 and α-synuclein and block their ability to form amyloid fibers. A long-term goal is to convert this β-hairpin peptide into a more drug-like molecule using chemical biology methods such as converting the α amino acids into β forms and using unnatural D stereoisomers of amino acids. The ultimate goal is to develop drugs that effectively combat the devastating neuronal diseases caused by aberrant protein aggregation. Support or Funding InformationThis material is based upon work supported in part by the U.S. Army Research Laboratory and the U.S. Army Research Office under grant number W911NF-13-1-0155. Bacterial Surface Display of Aggregation-Prone Proteins. (A) Aβ-42 peptide (B) α-synuclein

BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter.

BackgroundBacterial surface display technique enables the exogenous proteins or polypeptides displayed on the bacterial surface, while maintaining their relatively independent spatial structures and biological activities. The technique makes recombinant bacteria possess the expectant functions, subsequently, directly used for many applications. Many proteins could be used to achieve bacterial surface display, among them, autotransporter, a member of the type V secretion system of gram-negative bacteria, has been extensively studied because of its modular structure and apparent simplicity. However, autotransporter has not been widely used at present due to lack of a convenient genetic vector system. With our recently characterized autotransporter BrkA (Bordetella serum-resistance killing protein A) from Bordetella pertussis, we are aiming to develop a new autotransporter-based surface display system for potential wide application.ResultsHere, we construct a bacterial surface display system named as BrkAutoDisplay, based on the structure of autotransporter BrkA. BrkAutoDisplay is a convenient system to host exogenous genes. In our test, this system is good to efficiently display various proteins on the outer membrane surface of Escherichia coli, including green fluorescent protein (GFP), various enzymes and single chain antibody. Moreover, the displayed GFP possesses green fluorescence, the enzymes CotA, EstPc and PalA exhibit catalytic activity 0.12, 6.88 and 0.32 mU (per 5.2 × 108 living bacteria cells) respectively, and the single chain antibody fragment (scFv) can bind with its antigen strongly. Finally, we showed that C41(DE3) is a good strain of E. coli for the successful functionality of BrkAutoDisplay.ConclusionsWe designed a new bacterial display system called as BrkAutoDisplay and displayed various exogenous proteins on E. coli surface. Our results indicate that BrkAutoDisplay system is worthy of further study for industrial applications.

An efficient bacterial surface display system based on a novel outer membrane anchoring element from the Escherichia coli protein YiaT.

In a bacterial surface display system, the display of a successful recombinant protein is highly dependent on the choice of anchoring motif. In this study, we developed an efficient Escherichia coli display system using novel anchoring motifs derived from the protein YiaT. To determine the best surface-anchoring motif, full-length YiaT and two of its C-terminal truncated forms, cut at the R181 and R232 sites, were evaluated. Two industrial enzymes, a lipase from Pseudomonas fluorescens SIK W1 and an α-amylase from Bacillus subtilis, were used as the target proteins for display. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, immunofluorescence microscopy and whole-cell enzyme activity measurements confirmed the expression of the fusion proteins on the E. coli surface. Using YiaTR181 or YiaTR232 as the anchoring motif, the fusion proteins showed very high enzyme activities and did not exert any adverse effects on either cell growth or the outer membrane integrity. Additionally, these fusion proteins were suitable for displaying proteins of large molecular size in an active form. Compared with the previous anchoring motifs FadL and OprF, YiaTR181 and YiaTR232 had approximately 10-fold and 20-fold higher enzyme activities, respectively. These results suggest that YiaT can be used as an E. coli anchoring motif to efficiently display various enzymes; hence, this system could be employed in a variety of biotechnological and industrial applications.

Bacterial Surface Display of a Co-Factor Containing Enzyme, ω-Transaminase fromVibrio fluvialisUsing theBacillus subtilisSpore Display System

To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.

Ice nucleation protein as a bacterial surface display protein

Surface display technology can be defined as that phenotype (protein or peptide) which is linked to a genotype (DNA or RNA) through an appropriate anchoring motif. A bacterial surface display system is based on expressing recombinant proteins fused to sorting signals (anchoring motifs) that direct their incorporation on the cell surface.