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A low-cost affinity purification system using β-1,3-glucan recognition protein and curdlan beads

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Title: A low-cost affinity purification system using β-1,3-glucan recognition protein and curdlan beads
Authors: Horiuchi, Masataka Browse this author →KAKEN DB
Takahasi, Kiyohiro Browse this author
Kobashigawa, Yoshihiro Browse this author
Ochiai, Masanori Browse this author →KAKEN DB
Inagaki, Fuyuhiko Browse this author →KAKEN DB
Keywords: β-1,3-glucan recognition protein
affinity tag
curdlan
glutathione S-transferase
recombinant protein
Issue Date: Aug-2012
Publisher: Oxford University Press
Journal Title: Protein Engineering, Design and Selection (PEDS)
Volume: 25
Issue: 8
Start Page: 405
End Page: 413
Publisher DOI: 10.1093/protein/gzs028
Abstract: Silkworm β-1,3-glucan recognition protein (βGRP) tightly and specifically associates with β-1,3-glucan. We report here an affinity purification system named the 'GRP system', which uses the association between the β-1,3-glucan recognition domain of βGRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble β-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4-6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.
Type: article
URI: http://hdl.handle.net/2115/49735
Appears in Collections:生命科学院・先端生命科学研究院 (Graduate School of Life Science / Faculty of Advanced Life Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 稲垣 冬彦

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