Selection of ovine PrP high-producer subclones from a transfected epithelial cell line.
The hallmarks of prion diseases are the conversion of the normal prion into an abnormal protease resistant isoform and its brain accumulation. Purification of the native abnormal prion isoform for biochemical and biophysical studies has been hampered by poor recovery from brain tissue. An epithelial cell transfected with the ovine VRQ allele prion, called Rov9, has been used to select prion high-producer cells by flow cytometry. The representative clone 4 described here produced 6.2 microg of cellular prion protein per mg of total protein extract, representing 8- to 10-fold the amount produced by the Rov9 parental cells. After exposure to the scrapie agent (PG128/98), clone 4 produced 2.6 microg of abnormal isoform per mg of total protein. When infected clone 4 cell cultures were treated with tunicamycin, 80% of the abnormal isoform was deglycosylated. The infectivity of the prions produced in clone 4 cultures was confirmed in a mouse bioassay. Such high-producer clones represent new tools for producing large amounts of glycosylated and/or non-glycosylated PrP(Sc) and for a powerful screening of clinical samples' infectivity.
Falanga
Pierre B
PB
Unité de repliement et modélisation des protéines, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, France. pfalanga@pasteur.fr
Blom-Potar
Marie-Christine
MC
Bittoun
Patrick
P
Goldberg
Michel E
ME
Hontebeyrie
Mireille
M
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D018620:PrPSc Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods; D011817:Rabbits; D014162:Transfection / Q000379:methods
D018620:PrPSc Proteins