Applied Microbiology and Biotechnology

, Volume 98, Issue 12, pp 5677–5684 | Cite as

Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag

  • Mohamed A. A. Abdelhamid
  • Kei Motomura
  • Takeshi Ikeda
  • Takenori Ishida
  • Ryuichi Hirota
  • Akio Kuroda
Methods and protocols

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 Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results).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.

Keywords

Affinity purification Fusion tag Silica-binding peptide Small ubiquitin-like modifier Bacillus cereus CotB1 

Supplementary material

253_2014_5754_MOESM1_ESM.pdf (169 kb)
ESM 1(PDF 169 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Mohamed A. A. Abdelhamid
    • 1
  • Kei Motomura
    • 1
  • Takeshi Ikeda
    • 1
  • Takenori Ishida
    • 1
  • Ryuichi Hirota
    • 1
  • Akio Kuroda
    • 1
  1. 1.Department of Molecular Biotechnology, Graduate School of Advanced Sciences of MatterHiroshima UniversityHigashi-HiroshimaJapan

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