Probing the applicability of autotransporter based surface display with the EstA autotransporter of Pseudomonas stutzeri A15

Nicolay, Toon; Lemoine, Lynn; Lievens, Eva; Balzarini, Sam; Vanderleyden, Jos; Spaepen, Stijn

doi:10.1186/1475-2859-11-158

Cited by 15 publications

(12 citation statements)

References 53 publications

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“…The observed fitness cost for cells induced for NB-C-IgAP production, however, indicates that the explanation is meagre protein display, and points to LppOmpA as a more robust anchor in our system. While autotransporters have been reported as successful display anchors in many cases, our results are consistent with several studies where negative effects of autotransporter anchors were observed, with regards to cell viability [ 40 ], membrane integrity [ 41 ] and ultimately on surface display efficacy [ 8 ].…”

Section: Discussionsupporting

confidence: 92%

“…Bacterial surface display of enzymatically active proteins is a promising strategy to engineer whole-cell catalysts, enabling simplification of production procedures as well as downstream processes. Even though many surface display systems have been successfully employed since the first bacterial anchors were developed in the 1980s (reviewed in [ 4 ]), unpredictable, cargo-dependent effects hinder rational design and optimization, as exemplified by Nicolay et al [ 8 ] who reported failure to display a number of passenger domains with a previously characterised autotransporter.…”

Section: Discussionmentioning

confidence: 99%

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A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

et al. 2016

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BackgroundBacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay for evaluating and developing surface display systems is missing.ResultsUsing a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency.ConclusionsWe have developed an inexpensive and easy read-out assay for surface display using nanobody:GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies on the mechanism of protein transport to the surface of living cells, as well as the optimisation of applications in industrial biotech.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0474-y) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

et al. 2016

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“…ATs have been widely used as an elegant and efficient tool to display exogenous proteins and showed advantages for biotechnology and industrial (if scale up) applications [ 11 ]. However, not all target proteins can be successfully displayed [ 28 ] and till now there have been no universal rule to predict whether a target protein is suitable for AT-based surface display [ 9 ]. Furthermore, there is still lack of a convenient AT-based system for easier genetic operation and high throughput screening of target proteins.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

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“…Because ATs combine a relatively simple secretion mechanism with a flexible transport capacity, they have attracted attention as carriers for transport of heterologous proteins to the extracellular milieu (28,29). For this purpose, heterologous sequences have most often been fused directly to the C-terminal ␤-domain or to the N terminus of the passenger domain (27,30,31). We took a slightly different approach, capitalizing on the crystal structure of the Hbp passenger.…”

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confidence: 99%

Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Daleke-Schermerhorn¹,

Félix

Soprova³

et al. 2014

Appl Environ Microbiol

101

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iOuter membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ⌬tolR ⌬tolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

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Probing the applicability of autotransporter based surface display with the EstA autotransporter of Pseudomonas stutzeri A15

Cited by 15 publications

References 53 publications

A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

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

Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

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