title: A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.
abstract: 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.
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Sofie
authors.initials: S
authors.lastname: Wendel
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Emil C
authors.initials: EC
authors.lastname: Fischer
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Virginia
authors.initials: V
authors.lastname: Martínez
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Susanna
authors.initials: S
authors.lastname: Seppälä
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. morno@biosustain.dtu.dk.
authors.forename: Morten H H
authors.initials: MH
authors.lastname: Nørholm
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000276:immunology*; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D061905:Single-Domain Antibodies / Q000235:genetics / Q000378:metabolism*; D000069320:Type V Secretion Systems / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D000069320:Type V Secretion Systems; D049452:Green Fluorescent Proteins; C078167:OMPA outer membrane proteins; D002688:Chitinases
keywords: Autotransporter; Chitinase A; GFP; LppOmpA; Nanobody; Surface display; Whole-cell catalysis