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Oxidative refolding chromatography: folding of the scorpion toxin Cn5

Nature Biotechnology volume 17, pages 187–191 (1999)Cite this article

Abstract

We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components—GroEL minichaperone (191–345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl–prolyl isomerase—were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66–residue toxin, which has four disulfide bridges and a cis peptidyl–proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.

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Figure 1: Strategy for DsbA immobilization.
Figure 2: Overview of oxidative refolding chromatography.
Figure 3: Circular dichroism spectra of denatured and reduced Cn5 (––––) and refolded Cn5 (—).
Figure 4: Dose–response curve for native Cn5 toxin (open circles, see details under experimental protocol) and toxicity assay of refolded samples.

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Acknowledgements

M.M.A. was an EMBO fellow. This research was partially supported by a Howard Hughes Medical Institute grant (to L.D.P.). Fruitful discussions with Gilles Travé are also acknowledged.

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Authors and Affiliations

  1. Cambridge Centre for Protein Engineering and Cambridge University Chemical Laboratory, MRC Centre, Hills Rd., Cambridge, CB2 2QH, UK

    Myriam M. Altamirano & Alan R. Fersht

  2. Departamento de Bioquímica, Facultad de Medicina, UNAM, P.O. Box 70–159, México City, 04510, D. F, México

    Myriam M. Altamirano

  3. Department of Molecular Recognition and Structural Biology, Institute of Biotechnology, UNAM., P.O. Box 510–3, Cuernavaca, 62210, MOR, México

    Consuelo García & Lourival D. Possani

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  1. Myriam M. Altamirano
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Correspondence to Alan R. Fersht.

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Altamirano, M., García, C., Possani, L. et al. Oxidative refolding chromatography: folding of the scorpion toxin Cn5. Nat Biotechnol 17, 187–191 (1999). https://doi.org/10.1038/6192

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