Abstract
We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports.
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Acknowledgments
This work was supported in part by the Grant-in-Aid for Young Scientists (A) from the Japan Society for the Promotion of Science (No. 25712009 to T. Ikeda), the Industrial Technology Research Grant Program in 2009 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (No. 09C46130a to T. Ikeda), and a research grant from the Institute for Fermentation, Osaka (to T. Ikeda).
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Mohamed A. A. Abdelhamid and Kei Motomura contributed equally to this work.
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Abdelhamid, M.A.A., Motomura, K., Ikeda, T. et al. Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag. Appl Microbiol Biotechnol 98, 5677–5684 (2014). https://doi.org/10.1007/s00253-014-5754-z
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DOI: https://doi.org/10.1007/s00253-014-5754-z