Pilot study to determine the feasibility of producing protease-resistant prion protein fragments by random PCR mutagenesis.
We report the results of a pilot study to determine the feasibility of using PCR random mutagenesis and in vitro transcription/translation to produce protease resistant full-length or truncated ovine prion proteins (PrP). Using this approach, we show the novel production of protease resistant recombinant ovine prion protein fragments isolated from a panel of seventy randomly mutated ovine PrP protein molecules. Protease resistance of the proteinase K (PK) digested fragments was present de novo within physiological conditions without the need for template-assisted conversion to protease resistance or the requirement of reductants, denaturants or acid pH reported to date. Four of the mutant proteins were truncated at their C-termini and all of these gave rise to digestion products which were protease resistant at significant PK concentrations and exposure times. All other mutant proteins translated as full length molecules and gave rise to PK-resistant products which showed a variability in their proteinase digestion profiles. We discuss the relevance of these finding to current research.
Alzheimer's Disease and Prion Research Group, University of Cambridge School of Clinical Medicine, Department of Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, United Kingdom.
D V
DV
Dear
T J
TJ
Fitzmaurice
S
S
Garton
S J
SJ
Richards
D000818:Animals; D015153:Blotting, Western; D019286:Endopeptidase K / Q000378:metabolism*; D005240:Feasibility Studies; D008970:Molecular Weight; D016296:Mutagenesis; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010865:Pilot Projects; D016133:Polymerase Chain Reaction; D011328:Prions / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D012756:Sheep
D010446:Peptide Fragments; D011328:Prions; D019286:Endopeptidase K