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Updated: Jan 11, 2026
Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
Published on: December 12, 2017
A minimal slow dialysis method for refolding inclusion body proteins: Structural application to NEDD8 and its Q40E
Anna Makovska1, Erik Walinda2, Ryo Aoyama3
1Department of Biochemistry, Educational and Scientific Centre "Institute of Biology and Medicine", Taras Shevchenko National University of Kyiv, Ukraine; Laboratory of Integrative Molecular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Abstract:
NEDD8, a ubiquitin-like modifier essential for cullin-RING ligase regulation, is difficult to refold from Escherichia coli inclusion bodies due to aggregation during denaturant removal. Conventional refolding methods such as rapid dilution, one-step dialysis, and stepwise dialysis often produce poorly soluble proteins, limiting structural and functional analyses. To overcome this, we developed a simplified slow dialysis system using a single peristaltic pump to generate a gradual urea gradient over 96 h, enabling efficient refolding of NEDD8 under gentle conditions. Both wild-type and Q40E-mutant NEDD8, which mimics the bacteria-mediated deamidation observed during infection, refolded successfully into monomeric protein, as confirmed by size-exclusion chromatography. NMR spectra showed folded protein that matched reference assignments, demonstrating suitability for atomic level structural analysis. The method also enabled recovery of previously insoluble ISG15 mutants, highlighting its broad applicability for structural studies of diverse challenging proteins.
