On-column purification and refolding of recombinant bovine prion protein: using its octarepeat sequences as a natural affinity tag.
Prion protein has a key role in the occurrence of transmissible spongiform encephalopathy (TSE) and development of these diseases. Here, we provide a convenient procedure for on-column purification and refolding of the full-length mature bovine prion protein (bPrP) from Escherichia coli using immobilized metal (Ni) affinity chromatography, based on the metal-binding property of its unusual octarepeat sequences containing six tandem copies. Following extensive washing, the bPrP pellet was solubilized by guanidine hydrochloride and subjected to Ni-NTA agarose column. Purification and refolding were achieved by stepwise gradient washing with reduced guanidine hydrochloride concentrations. Triton X-100 and beta-mercaptoethanol were required in this rapid refolding process. The isolated prion protein was identified by monoclonal antibodies and its integrity was monitored by mass spectroscopy. Its correct folding was confirmed from circular dichroism (CD) experiments. Moreover, thioflavin T-binding assay showed that the recombinant bPrP could be transformed into amyloid fiber structures like that of the infectious prion isoform PrP(sc).
Yin
Shao-Man
SM
Department of Molecular Virology, Institute of Microbiology, Chinese Academy of Science, P.O. Box 2714, Beijing 100080, PR China.
Zheng
Yi
Y
Tien
Po
P
D000682:Amyloid / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004926:Escherichia coli; D009532:Nickel / Q000378:metabolism; D011328:Prions / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D000682:Amyloid; D011328:Prions; D011993:Recombinant Fusion Proteins; D009532:Nickel