On-column refolding of recombinant human interleukin-4 from inclusion bodies.
Interleukin-4 (IL4) is a multifunctional cytokine which plays a key role in the immune system. Several antagonists/agonists of IL4 are reported through mutagenesis studies, but their solution structural studies using nuclear magnetic resonance (NMR) spectroscopy are hindered as milligram quantities of isotopically labeled protein are required for structural refinements. In this work, a His-tagged recombinant form of human IL4 was overexpressed in Escherichia coli under the control of a T7 promoter. The resulting inclusion bodies were separated from cellular debris by centrifugation and solubilized by 6M guanidine-HCl in the presence of reducing agents. The denatured IL4 was immobilized on Ni2+-fractogel beads and refolded in a single chromatographic step by gradual removal of denaturant. This protocol yielded 15-20 mg of isotope-enriched protein from 1L of culture grown in minimal medium. The refolded protein was highly pure and was correctly folded as judged by its two-dimensional NMR spectrum. To show the successful application of this refolding protocol to IL4 variants, 15N-labeled Y124D-IL4 was also prepared and its first two-dimensional NMR spectrum was presented.
Razeghifard
M Reza
MR
Department of Human Biological Chemistry and Genetics, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555, USA. razeghifard@rsbs.anu.edu.au
D000595:Amino Acid Sequence; D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D015847:Interleukin-4 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D015847:Interleukin-4