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 usin g PCR random mutagenesis and in vitro transcription/translation to produce protease resistant full-length or truncated ovine prion proteins (PrP). Usi ng this approach, we show the novel production of protease resistant recomb inant ovine prion protein fragments isolated from a panel of seventy random ly mutated ovine PrP protein molecules. Protease resistance of the proteina se K (PK) digested fragments was present de novo within physiological condi tions without the need for template-assisted conversion to protease resista nce or the requirement of reductants, denaturants or acid pH reported to da te. Four of the mutant proteins were truncated at their C-termini and all o f these gave rise to digestion products which were protease resistant at si gnificant PK concentrations and exposure times. All other mutant proteins t ranslated as full length molecules and gave rise to PK-resistant products w hich showed a variability in their proteinase digestion profiles. We discus s the relevance of these finding to current research. (C) 2001 Academic Pre ss.