Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation.
A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.
Jones
Michael
M
National CJD Surveillance Unit, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. mike.jones@snbts.csa.scot.nhs.uk
Head
Mark W
MW
Connolly
John G
JG
Farquhar
Christine F
CF
Hornsey
Valerie S
VS
Pepper
Duncan S
DS
MacGregor
Ian R
IR
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000737:chemistry / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008970:Molecular Weight; D017830:Octoxynol / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D011328:Prions / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D020836:Protein Structure, Quaternary
D007476:Ionophores; D011328:Prions; D003300:Copper; D017830:Octoxynol; D002118:Calcium