What is it about?
NEDD8 is a small protein that helps control cell division, embryonic development, and DNA repair. But when it is expressed in bacterial cells, it forms insoluble aggregates, making it difficult to purify and study. We developed a faster, simpler method of On-Column refolding, which gives a fourfold higher yield of correctly folded protein as compared with earlier urea-based approaches.
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Why is it important?
NEDD8 is a ubiquitin-like modifier that regulates cullin-RING E3 ligases and, through them, the cell cycle, embryonic development, and the DNA damage response, so obtaining pure, properly folded protein is essential for biochemical and structural studies. Despite sharing over 60% sequence identity and a similar fold with highly soluble ubiquitin, recombinant NEDD8 expressed in E. coli accumulates in inclusion bodies. Conventional refolding by stepwise dialysis causes aggregation as the denaturant is gradually diluted, leaving low yields of native monomer. We instead bound His-tagged NEDD8 to a nickel-affinity column under denaturing conditions and removed the denaturant rapidly on the column, minimizing the time spent at intermediate denaturant concentrations. Circular dichroism and NMR spectroscopy confirmed correct secondary and tertiary structure. The method works in both rich and isotope-enriched minimal media, reducing protein loss and cost, and may serve as a starting point for other small, aggregation-prone proteins, though case-by-case optimization is needed.
Perspectives
As someone who works with biophysical studies of proteins, I know how frustrating it is when a protein you need in large amounts ends up as clumps, especially in case of NMR when every preparation uses expensive isotope-labeled media. NEDD8 was a good example, it is so similar to ubiquitin, yet so much harder to produce. What we found most useful was a simple change in thinking; instead of slowly removing the denaturant, remove it quickly while the protein is held on the column. If you are struggling with a small, aggregation-prone protein, this approach may be worth trying. I hope it saves other labs time and material.
GS Lab
Central University of Rajasthan
Read the Original
This page is a summary of: Optimized Strategy for Expression, On-Column Refolding, and Purification of NEDD8 Protein, Protein & Peptide Letters, February 2026, Bentham Science Publishers,
DOI: 10.2174/0109298665422034251021111813.
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