Large-scale production, purification and refolding of the full-length cellular prion protein from Syrian golden hamster in Escherichia coli using the glutathione S-transferase-fusion system.
Until quite recently, high-level expression of full-length cellular prion protein (Prp(c)) in bacterial cells was not possible. We describe here the effective purification of mature Syrian golden hamster PrPc (residues 23-231) as a C-terminal fusion to glutathione S-transferase (GST) from inclusion bodies expressed in Escherichia coli. Purification of the denatured fusion protein was simplified greatly by the introduction of a C-terminal histidine anchor, leading to 255 mg pure GST-PrPc-His6/l bacterial broth, which could be refolded easily by dilution in 20 mM Tris, 5 mM dithiothreitol, 1 mM EDTA, pH 9.0. Refolding was monitored by following GST activity. Mature Syrian hamster PrPc (residues 23-231) was released from the refolded fusion protein by thrombin digestion, yielding 73 mg homogeneous protein/l bacterial culture after purification. The recombinant protein was identified by monoclonal antibodies, Edman sequencing and matrix-assisted laser-desorption/ionization MS. Correct folding was confirmed by near-ultraviolet circular dichroism spectroscopy. Samples resulting from different purification steps were sensitive to proteinase K digestion and showed no signs of infectivity in animal experiments, demonstrating that the PrPc produced is identical with the cellular isoform. The presented purification procedure should prove useful for the production of other GST-fusion proteins.
Völkel
D
D
Department of Structure Research, GBF-National Research Centre for Biotechnology, Braunschweig, Germany. dvo@gbf-braunschweig.de
Blankenfeldt
W
W
Schomburg
D
D
D000818:Animals; D002942:Circular Dichroism; D006224:Cricetinae; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D018621:PrPC Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D012995:Solubility; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism
D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D013917:Thrombin; D019286:Endopeptidase K