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Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure

¹ Department of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon, Korea
² Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA
³ Integrity Biosolution, Camarillo, California 93012, USA
⁴ Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, USA

(RECEIVED August 28, 2005; FINAL REVISION November 4, 2005; ACCEPTED November 7, 2005)

High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three Gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of ~42%–58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (~70%–100%) to achieve refolding yields of ~55%–78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.

Keywords: high hydrostatic pressure; refolding; inclusion body; solutes; redox-shuffling agent; arginine

Abbreviations: HHP, high hydrostatic pressure • IB, inclusion body • SEC, size-exclusion chromatography • GNBP, Gram-negative binding protein • DTT, dithiothreitol • GSSG, oxidized glutathione

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051813506.

Reprint requests to: Yong-Sung Kim, Ph.D., Department of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon, Korea; e-mail: kimys{at}ajou.ac.kr.; fax: 82-31-219-2394.

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