Optimized overproduction, purification, characterization and high-pressure sensitivity of the prion protein in the native (PrP(C)-like) or amyloid (PrP(Sc)-like) conformation.
Overproduction and purification of the prion protein is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. We have developed a method for the effective cloning, overexpression in Escherichia coli and purification to homogeneity of Syrian golden hamster prion protein (SHaPrP(90-231)). A high level of overexpression, resulting in the formation of inclusion bodies, was obtained under the control of the T7-inducible promoter of the pET15b plasmid. The protein required denaturation, reduction and refolding steps to become soluble and attain its native conformation. Purification was carried out by differential centrifugation, gel filtration and reverse phase chromatography. An improved cysteine oxidation protocol using oxidized glutathione under denaturing conditions, resulted in the recovery of a higher yield of chromatographically pure protein. About 10 mg of PrP protein per liter of bacterial culture was obtained. The recombinant protein was identified by monoclonal antibodies and its integrity was confirmed by electrospray mass spectrometry (ES/MS), whereas correct folding was assessed by circular dichroism (CD) spectroscopy. This protein had the structural characteristics of PrP(C) and could be converted to an amyloid structure sharing biophysical and biochemical properties of the pathologic form (PrP(Sc)). The sensitivity of these two forms to high pressure was investigated. We demonstrate the potential of using pressure as a thermodynamic parameter to rescue trapped aggregated prion conformations into a soluble state, and to explore new conformational coordinates of the prion protein conformational landscape.
Alvarez-Martinez
Maria Teresa
MT
INSERM U431, CC100, Dept Biologie Sante, Université de Montpellier 2, Place Eugène Bataillon, F-34095 Montpellier Cedex 5, France.
Torrent
Joan
J
Lange
Reinhard
R
Verdier
Jean-Michel
JM
Balny
Claude
C
Liautard
Jean-Pierre
JP
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D010957:Plasmids; D018621:PrPC Proteins / Q000737:chemistry / Q000235:genetics; D018620:PrPSc Proteins / Q000737:chemistry / Q000235:genetics; D011312:Pressure; D011328:Prions / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
D018621:PrPC Proteins; D018620:PrPSc Proteins; D011328:Prions; D020033:Protein Isoforms; D011994:Recombinant Proteins