Display of native proteins on Bacillus subtilis spores

• Published in: FEMS microbiology letters 2014-09, Vol.358 (2), p.209-217
• Main Authors: Pan, Jae-Gu, Choi, Soo-Keun, Jung, Heung-Chae, Kim, Eui-Joong
• Format: Article
• Language: English
• Subjects: Bacillus; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacterial Proteins - genetics; Bacteriology; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Bioremediation; Biosensors; carboxymethylcellulase; Cell Surface Display Techniques; Cellulase - genetics; Cellulase - metabolism; Endotoxins - genetics; Enzymatic activity; Enzymes; Flow Cytometry; Gene Expression; Hemolysin Proteins - genetics; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microbiology; Microscopy, Immunoelectron; native display; Promoter Regions, Genetic; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; spore; Spores - genetics; Spores - metabolism; β‐galactosidase
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/1574-6968.12558

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 σE and σK 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. Target proteins are displayed on the surface of Bacillus spores in their native forms, obviating the need to make fusion proteins to anchoring motifs. Graphical Abstract Figure. Target proteins are displayed on the surface of Bacillus spores in their native forms, obviating the need to make fusion proteins to anchoring motifs.