Journal
MICROBIAL CELL FACTORIES
Volume 15, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s12934-016-0474-y
Keywords
Surface display; Nanobody; GFP; Chitinase A; LppOmpA; Autotransporter; Whole-cell catalysis
Categories
Funding
- Novo Nordisk Foundation
- People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme, BACTORY [317058]
- VILLUM Foundation's Young Investigator Programme [VKR023128]
- Novo Scholarship Programme
- NNF Center for Biosustainability [Microbial Evolution & Synthetic Bio] Funding Source: researchfish
- Novo Nordisk Fonden [NNF10CC1016517] Funding Source: researchfish
- Villum Fonden [00007277] Funding Source: researchfish
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Background: Bacterial 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. Results: Using 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. Conclusions: We 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.
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