Enhanced display of lipase on the Escherichia coli cell surface, based on transcriptome analysis.
Ontology highlight
ABSTRACT:
SUBMITTER: Baek JH
PROVIDER: S-EPMC2812984 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50 degrees C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70 degrees C, and retained over 90% of the full activity after incubation at 50 degrees C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.
| S-EPMC520891 | biostudies-literature
Project description:BACKGROUND: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the ?-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. RESULTS: Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. CONCLUSION: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.
| S-EPMC3094208 | biostudies-literature
Project description:BackgroundThe display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins.ResultsIn this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp'OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp'OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy.ConclusionsTo our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp'OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.
| S-EPMC7853708 | biostudies-literature
Project description:Concerns over the increasing emergence of antibiotic-resistant pathogenic microorganisms due to the overuse of antibiotics and the lack of effective antibiotics for livestock have prompted efforts to develop alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) with a broad-spectrum activity and rapid killing, along with little opportunity for the development of resistance, represent one of the promising novel alternatives. Their high production cost and cytotoxicity, however, limit the use of AMPs as effective antibiotic agents to livestock. To overcome these problems, we developed potent antimicrobial Escherichia coli displaying multimeric AMPs on the cell surface so that the AMP multimers can be converted into active AMP monomers by the pepsin in the stomach of livestock. Buf IIIb, a strong AMP without cytotoxicity, was expressed on the surface of E. coli as Lpp-OmpA-fused tandem multimers with a pepsin substrate residue, leucine, at the C-terminus of each monomer. The AMP multimers were successfully converted into active AMPs upon pepsin cleavage, and the liberated Buf IIIb-L monomers inhibited the growth of two major oral infectious pathogens of livestock, Salmonella enteritidis and Listeria monocytogenes. Live antimicrobial microorganisms developed in this study may represent the most effective means of providing potent AMPs to livestock, and have a great impact on controlling over pathogenic microorganisms in the livestock production.
| S-EPMC3597565 | biostudies-literature
Project description:Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter ?-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture.
| S-EPMC4337897 | biostudies-literature
Project description:Mussels attach to virtually all types of inorganic and organic surfaces in aqueous environments, and catecholamines composed of 3,4-dihydroxy-l-phenylalanine (DOPA), lysine, and histidine in mussel adhesive proteins play a key role in the robust adhesion. DOPA is an unusual catecholic amino acid, and its side chain is called catechol. In this study, we displayed the adhesive moiety of DOPA-histidine on Escherichia coli surfaces using outer membrane protein W as an anchoring motif for the first time. Localization of catecholamines on the cell surface was confirmed by Western blot and immunofluorescence microscopy. Furthermore, cell-to-cell cohesion (i.e., cellular aggregation) induced by the displayed catecholamine and synthesis of gold nanoparticles on the cell surface support functional display of adhesive catecholamines. The engineered E. coli exhibited significant adhesion onto various material surfaces, including silica and glass microparticles, gold, titanium, silicon, poly(ethylene terephthalate), poly(urethane), and poly(dimethylsiloxane). The uniqueness of this approach utilizing the engineered sticky E. coli is that no chemistry for cell attachment are necessary, and the ability of spontaneous E. coli attachment allows one to immobilize the cells on challenging material surfaces such as synthetic polymers. Therefore, we envision that mussel-inspired catecholamine yielded sticky E. coli that can be used as a new type of engineered microbe for various emerging fields, such as whole living cell attachment on versatile material surfaces, cell-to-cell communication systems, and many others.
| S-EPMC3911018 | biostudies-literature
Project description:A new system designed for cell surface display of recombinant proteins on Escherichia coli has been evaluated for expression of eukaryotic viral proteins. Human immunodeficiency virus type 1 (HIV-1) gp120 was fused to the C terminus of ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, fluorescence-activated cell-sorting analysis, whole-cell enzyme-linked immunosorbent assay, and ice nucleation activity assay confirmed the successful expression of HIV-1 gp120 on the surface of Escherichia coli. This study shows that the INP system can be used for the expression of eukaryotic viral proteins. There is also a possibility that the INP system can be used as an AIDS diagnostic system, an oral vaccine delivery system, and an expression system for various heterologous higher-molecular-weight proteins.
| S-EPMC95715 | biostudies-literature
Project description:In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control cells indicating that mCherry was displayed on the cells. Expression of mCherry on cells of Y. lipolytica was confirmed by both fluorescent microscopy and flow cytometry. Furthermore, SDS-PAGE analysis and matrix-assisted laser desorption/ionization (MALDI)-time-of (TOF)-mass spectrometry (MS) peptide mass fingerprinting (PMF) confirmed that the protein cleaved from the yeast cells using enterokinase was mCherry. Efficient cleavage of mCherry reported in this work offers an alternative purification method for displayed heterologous proteins on Y. lipolytica cells using the plasmid constructed in this study. The developed displaying system offers great potential for industrial production and purification of heterologous proteins at low cost.
| S-EPMC3441212 | biostudies-literature
Project description:BackgroundFor industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.ResultsTwo Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the alpha-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40 degrees C at pH8.0, they retained over 90% activity after incubation at 60 degrees C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.ConclusionThe LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.
| S-EPMC2267459 | biostudies-literature
Project description:A cell surface displayed system in Pichia pastoris GS115 was developed by using GCW61, a glycosylphosphatidylinositol-modified cell wall protein from P. pastoris, as the anchor protein. Thermomyces lanuginosus lipase (TLL) was successfully displayed on the P. pastoris cell wall by fusing GCW61 gene with TLL2 gene (NCBI Accession: O59952) that was optimized with codon bias and synthesized. Cell surface displayed TLL2 was confirmed by the immunofluorescence microscopy. Flask fermentation was performed for 144 h with lipase activity up to 1964.76 U/g. Enzymatic properties of cell surface displayed TLL2 were also investigated. Displayed TLL2 occurred the maximum activity at pH 9 and 55°C and demonstrated characteristics of wide thermal adaptability and alkaline pH resistance. The optimum substrate was p-nitrophenyl hexanoate. Bivalent metal ions Ca2+, Mn2+, and Zn2+ had the activation effect on displayed TLL2, while Cu2+, Fe2+, Fe3+, K+, Li+, Na+, and Co2+ ions had the inhibitory effect on it. Since cell surface displayed TLL2 required less purification steps compared with free enzyme and showed high enzyme activities, it would be able to be further applied in various potential applications.
| S-EPMC7656992 | biostudies-literature