Green fluorescent protein as a reporter of prion protein folding.
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.


CONCLUSIONS
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.
Vasiljevic
Snezana
S
School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK. s.vasiljevic@rdg.ac.uk
Ren
Junyuan
J
Yao
YongXiu
Y
Dalton
Kevin
K
Adamson
Catherine S
CS
Jones
Ian M
IM
D000818:Animals; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018621:PrPC Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003300:Copper