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High-Level Expression and Characterization of a Purified 142-Residue Polypeptide of the Prion Protein

1996; American Chemical Society; Volume: 35; Issue: 17 Linguagem: Inglês

10.1021/bi952965e

1943-295X

Ingrid Mehlhorn, Darlene Groth, Johannes Stöckel, Barbara Moffat, Dorothea Reilly, Daniel G. Yansura, W. Scott Willett, Michael A. Baldwin, Robert J. Fletterick, Fred E. Cohen, Richard Vandlen, Dennis J. Henner, Stanley B. Prusiner,

Trace Elements in Health

The major, and possibly only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27−30, a protein of ∼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 truncated 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 expression was obtained for a truncated SHaPrP containing residues 90−231, which correspond to the sequence of PrP 27−30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHCl, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was ∼50%, and from each liter of E. coli culture, ∼50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high β-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 residues of this polypeptide produced a soluble protein with a high α-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occuring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that PrP can adopt.