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The work described here was supported by the National Institutes of Health GM 62412 - PowerPoint PPT Presentation


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On-column Chemical Refolding Natalia Oganesyan and Rosalind Kim Lawrence Berkeley National Laboratory PPCW, March 29-31, 2004. The work described here was supported by the National Institutes of Health GM 62412 . IB in 8 M urea Dilution into a large volume of buffer with detergent

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    1. On-columnChemical Refolding Natalia Oganesyan and Rosalind Kim Lawrence Berkeley National Laboratory PPCW, March 29-31, 2004 The work described here was supported by the National Institutes of Health GM 62412

    2. IB in 8 M urea Dilution into a large volume of buffer with detergent Dilution with -cyclodextrin buffer Filtration (0.2 μ) Concentration IB in 8 M urea Binding to Ni-NTA Wash with buffer containing detergent Wash with buffer containing -cyclodextrin Elution with imidazole Chemical Refolding of protein inclusion bodies (IB) Daugherty et al., 1998 JBC, vol. 273, 33961-71 BSGC on-column refolding method for non-membrane proteins Time required 2-3 days 20 hrs

    3. Detergent mini-refolding screen

    4. BSGC refolding protocol • Solubilization of IB in 8 M urea or 6 M GuHCl • Overnight binding to Ni-NTA (Qiagen) • Column wash with urea buffer and 20 mM imidazole • Wash with 0.6 mM Triton X-100 or DDM under native conditions • Wash with 5 mM -cyclodextrin • Wash with 0.5 M NaCl • Elution with 400 mM imidazole • IEX or SEC

    5. Summary of refolded BSGC targets Targets MW, kDa% “Refolded” *Crystallized • 1084B 40 40% yes • 1349B 20 100% 2.8 A data • 1105B 70 100% 3.2 A data/solved • 1113B 19 100% yes • 1049B 36 50% yes • 1277B 44 100% yes • 1089B 16.7 100% no • 3 targets failed to refold *% target eluted / % target loaded

    6. SeMet Crystals of 1105B Refolded 1105B Soluble 1105B 0.1 M Tris-HCl, pH 8.5, 0.2 MLi2SO4,30%(w/v) PEG 4K 0.1 M HEPES, pH 7.5, 1.5 M Li2SO4

    7. Crystal structure of 1105B Refolded Soluble 3.2 Å P32 2.8 Å P43 2 molecules per a.u. 6 molecules per a.u. V.Oganesyan et al. Manuscript in preparation

    8. Summary • On-column chemical refolding is fast and provides an easy way to refold various His-tagged proteins. • Mini-purification screen allows selection of the most efficient detergent. It can be formulated as a high-throughput method. • Seven out of ten tested proteins have been refolded by this method. • Effective refolding provides enough soluble protein for crystallization trials.