Green fluorescent protein as a reporter of prion protein folding

Vasiljevic, S., Ren, J. Y., Yao, Y. X., Dalton, K., Adamson, C. S. and Jones, I. M. ORCID: https://orcid.org/0000-0002-7738-2516 (2006) Green fluorescent protein as a reporter of prion protein folding. Virology Journal, 3. p. 9. ISSN 1743-422X doi: 10.1186/1743-422x-3-59

Abstract/Summary

Background: The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP), as a method to examine the refolding of the amino terminal domain of mouse prion protein. Results: Fusion proteins of PrPc and GFP were expressed at high level in E. coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E. coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence. Conclusion: Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/10243
Item Type	Article
Refereed	Yes
Divisions	Life Sciences > School of Biological Sciences
Uncontrolled Keywords	OXIDATIVE STRESS, BINDING-SITES, N-TERMINUS, IN-VIVO, COPPER-BINDING, MISFOLDING DISEASES, DIRECTED EVOLUTION, INFECTION IMPAIRS, CELLULAR-RESPONSE, SCRAPIE
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