HIGH-LEVEL EXPRESSION AND CHARACTERIZATION OF A PURIFIED 142-RESIDUE POLYPEPTIDE OF THE PRION PROTEIN

The major, and possibly only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc i s PrP 27-30, a protein of similar to 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C-terminally trun cated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum ex pression was obtained for a truncated SHaPrP containing residues 90-23 1, which correspond to the sequence of PrP 27-30; disruption of the ba cteria using a microfluidizer produced the highest yields of this prot ein designated rPrP. After solubilization of rPrP in 8 M GdnHCl, it wa s purified by size exclusion chromatography and reversed phase chromat ography. During purification the recovery was similar to 50%, and from each liter of E. coli culture, similar to 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-term inal region was less successful and was not pursued further. The prima ry structure of rPrP was verified by Edman sequencing and mass spectro metry, and secondary structure determined by circular dichroism and Fo urier transform infrared spectroscopy. When rPrP was purified under re ducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values >7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys resi dues of this polypeptide produced a soluble protein with a high ct-hel ical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the natura lly occuring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertia ry structures that PrP can adopt.