title: Display of native proteins on Bacillus subtilis spores.
abstract: In principle, protein display is enabled by fusing target proteins to naturally secreted, surface-anchored protein motifs. In this work, we developed a method of native protein display on the Bacillus spore surface that obviates the need to construct fusion proteins to display a motif. Spore coat proteins are expressed in the mother cell compartment and are subsequently assembled and deposited on the surface of spores. Therefore, target proteins overexpressed in the mother cell compartment during the late sporulation phase were expected to be targeted and displayed on the spore surface. As a proof of principle, we demonstrated the display of carboxymethylcellulase (CMCase) in its native form on the spore surface. The target protein, CMCase, was expressed under the control of the cry1Aa promoter, which is controlled by σ(E) and σ(K) and is expressed in the mother cell compartment. The correct display was confirmed using enzyme activity assays, flow cytometry, and immunogold electron microscopy. In addition, we demonstrated the display of a β-galactosidase tetramer and confirmed its correct display using enzyme activity assays and protein characterization. This native protein display system, combined with the robust nature of Bacillus spores, will broaden the range of displayable target proteins. Consequently, the applications of display technology will be expanded, including high-throughput screening, vaccines, biosensors, biocatalysis, bioremediation, and other innovative bioprocesses.
authors.affiliation: Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.
authors.forename: Jae-Gu
authors.initials: JG
authors.lastname: Pan
authors.forename: Soo-Keun
authors.initials: SK
authors.lastname: Choi
authors.forename: Heung-Chae
authors.initials: HC
authors.lastname: Jung
authors.forename: Eui-Joong
authors.initials: EJ
authors.lastname: Kim
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics; D061505:Cell Surface Display Techniques; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013170:Spores / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D001616:beta-Galactosidase; D002480:Cellulase; C019826:carboxymethylcellulase
keywords: Bacillus; carboxymethylcellulase; native display; spore; β-galactosidase