Optimized Strategy for Expression, On-Column Refolding, and Purification of NEDD8 Protein

Ubiquitin and the Ubls family are known for their high solubility and excellent expression profiles in recombinant systems. In contrast, Neural Precursor Cell Expressed, Developmentally Down-Regulated 8 (NEDD8) is a ubiquitin-like modifier that shares more than 60% sequence identity with ubiquitin and exhibits a similar structural fold. NEDD8 primarily functions by modifying the cullin subunits of cullin-RING E3 ligases, thereby playing a critical role in regulating the cell cycle, embryonic development, and DNA repair processes, particularly by localizing to sites of DNA damage. Despite its structural and functional similarity to highly soluble ubiquitin family proteins, recombinant NEDD8 is predominantly expressed in inclusion bodies, making its purification challenging.

Traditional refolding and purification strategies using 6M urea have proven inefficientin recovering properly folded and functional protein. In this study, we present a streamlined, high- - yield method for purifying NEDD8 based on on-column refolding using a 6xHis tag in combina-- tion with nickel-affinity chromatography, followed by size exclusion chromatography for further purification. We applied an on-column refolding method for NEDD8, eliminating dialysis-associated losses and yielding well-folded protein. The approach is effective for small proteins but limited by size, hydrophobicity, and charge-related aggregation risks. Broader applicability requires case-specific optimization to ensure correct folding and structural fidelity across diverse proteins.

The structural integrity and correct folding of the purified NEDD8 were confirmed through both nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy, validating the effectiveness of the method for producing biologically relevant, properly folded protein.

Our results demonstrate that this on-column refolding approach significantly improves the yield and refolding efficiency of NEDD8 compared to previous urea-based methods.