Skip to content
2000
image of Optimized Strategy for Expression, On-Column Refolding, and Purification of NEDD8 Protein

Abstract

Introduction

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.

Methods

Traditional refolding and purification strategies using 6M urea have proven inefficient in 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 combination with nickel-affinity chromatography, followed by size exclusion chromatography for further purification.

Results

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.

Limitation of the Study

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.

Conclusion

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

© 2026 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/ppl/10.2174/0109298665422034251021111813
2026-01-22
2026-02-23

  • From This Site

    /content/journals/ppl/10.2174/0109298665422034251021111813
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. Herhaus L. Dikic I. Expanding the ubiquitin code through post-translational modification. EMBO Rep. 2015 16 9 1071 1083 10.15252/embr.201540891 26268526
    [Google Scholar]
  2. Wingfield P.T. Palmer I. Liang S.M. Folding and purification of insoluble (inclusion body) proteins from Escherichia coli. Curr Protoc Protein Sci 2014 78 6.5.1 6.5.30 10.1002/0471140864.ps0605s78 25367010
    [Google Scholar]
  3. McConnell M.J. Pachón J. Expression, purification, and refolding of biologically active Acinetobacter baumannii OmpA from Escherichia coli inclusion bodies. Protein Expr. Purif. 2011 77 1 98 103 10.1016/j.pep.2010.11.019 21130880
    [Google Scholar]
  4. Fischer B.E. Renaturation of recombinant proteins produced as inclusion bodies. Biotechnol. Adv. 1994 12 1 89 101 10.1016/0734‑9750(94)90292‑5 14547821
    [Google Scholar]
  5. Singhvi P. Panda A.K. Solubilization and refolding of inclusion body proteins. Methods Mol Biol 2022 2406 371 387 10.1007/978‑1‑0716‑1859‑2_22 35089569
    [Google Scholar]
  6. Kopp J. Spadiut O. Inclusion bodies: Status quo and perspectives. Methods Mol Biol 2023 2617 1 13 10.1007/978‑1‑0716‑2930‑7_1 36656513
    [Google Scholar]
  7. Tsumoto K. Ejima D. Kumagai I. Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein Expr. Purif. 2003 28 1 1 8 10.1016/S1046‑5928(02)00641‑1 12651100
    [Google Scholar]
  8. Yamaguchi H. Miyazaki M. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules 2014 4 1 235 251 10.3390/biom4010235 24970214
    [Google Scholar]
  9. Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein. J. Biol. Chem. 1997 272 45 28557 28562 10.1074/jbc.272.45.28557 9353319
    [Google Scholar]
  10. Zou T. Zhang J. Diverse and pivotal roles of neddylation in metabolism and immunity. FEBS J. 2021 288 13 3884 3912 10.1111/febs.15584 33025631
    [Google Scholar]
  11. Malakhov M.P. Mattern M.R. Malakhova O.A. Drinker M. Weeks S.D. Butt T.R. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J. Struct. Funct. Genomics 2004 5 1-2 75 86 10.1023/B:JSFG.0000029237.70316.52 15263846
    [Google Scholar]
  12. Yau R. Rape M. The increasing complexity of the ubiquitin code. Nat. Cell Biol. 2016 18 6 579 586 10.1038/ncb3358 27230526
    [Google Scholar]
  13. Dil Kuazi A. Kito K. Abe Y. Shin R.W. Kamitani T. Ueda N. Nedd8 protein is involved in ubiquitinated inclusion bodies. J Pathol 2003 199 2 259 266 10.1002/path.1283 12533840
    [Google Scholar]
  14. Streich F.C. Jr Lima C.D. Structural and functional insights to ubiquitin-like protein conjugation. Annu. Rev. Biophys. 2014 43 1 357 379 10.1146/annurev‑biophys‑051013‑022958 24773014
    [Google Scholar]
  15. Melchior F. SUMO--nonclassical ubiquitin. Annu. Rev. Cell Dev. Biol. 2000 16 1 591 626 10.1146/annurev.cellbio.16.1.591 11031248
    [Google Scholar]
  16. Kerscher O. Felberbaum R. Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 2006 22 1 159 180 10.1146/annurev.cellbio.22.010605.093503 16753028
    [Google Scholar]
  17. Dye B.T. Schulman B.A. Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu. Rev. Biophys. Biomol. Struct. 2007 36 1 131 150 10.1146/annurev.biophys.36.040306.132820 17477837
    [Google Scholar]
  18. Greenfield N.J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2006 1 6 2876 2890 10.1038/nprot.2006.202 17406547
    [Google Scholar]
  19. Panavas T. Sanders C. Butt T.R. Sumo fusion technology for enhanced protein production in prokaryotic and eukaryotic expression systems. Methods Mol Biol 2009 497 303 317 10.1007/978‑1‑59745‑566‑4_20 19107426
    [Google Scholar]
  20. Wang Z. Li H. Guan W. Ling H. Wang Z. Mu T. Shuler F.D. Fang X. Human SUMO fusion systems enhance protein expression and solubility. Protein Expr. Purif. 2010 73 2 203 208 10.1016/j.pep.2010.05.001 20457256
    [Google Scholar]
  21. Butt T.R. Edavettal S.C. Hall J.P. Mattern M.R. SUMO fusion technology for difficult-to-express proteins. Protein Expr. Purif. 2005 43 1 1 9 10.1016/j.pep.2005.03.016 16084395
    [Google Scholar]
  22. Hochstrasser M. Origin and function of ubiquitin-like proteins. Nature 2009 458 7237 422 429 10.1038/nature07958 19325621
    [Google Scholar]
  23. Santonico E. Old and New Concepts in Ubiquitin and NEDD8 Recognition. Biomolecules 2020 10 4 566 10.3390/biom10040566 32272761
    [Google Scholar]
  24. Whitby F.G. Xia G. Pickart C.M. Hill C.P. Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes. J. Biol. Chem. 1998 273 52 34983 34991 10.1074/jbc.273.52.34983 9857030
    [Google Scholar]
  25. Cappadocia L. Lima C.D. Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism. Chem. Rev. 2018 118 3 889 918 10.1021/acs.chemrev.6b00737 28234446
    [Google Scholar]
/content/journals/ppl/10.2174/0109298665422034251021111813
Loading
Optimized Strategy for Expression, On-Column Refolding, and Purification of NEDD8 Protein
Bentham Science Publishers ; https://doi.org/10.2174/0109298665422034251021111813
/content/journals/ppl/10.2174/0109298665422034251021111813
/content/journals/ppl/10.2174/0109298665422034251021111813
Loading

Data & Media loading...

Supplements

Supplementary material is available on the publisher’s website along with the published article.

file format download Download

  • Article Type:     Research Article
Keywords: DNA damage ; refolding ; CD spectroscopy ; NMR spectroscopy ; NEDD8 ; Ubiquitin

Most Cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test