Abstract:
:An efficient method is presented for the production of intact mammalian prion proteins and partial sequences thereof. As an illustration we describe the production of polypeptides comprising residues 23-231, 81-231, 90-231 and 121-231 of the human prion protein (hPrP). Polypeptides were expressed as histidine tail fusion proteins into inclusion bodies in the cytoplasm of Escherichia coli and refolded and oxidized while N-terminally immobilized on a nickel-NTA agarose resin. This 'high-affinity column refolding' facilitates the preparation of prion proteins by preventing protein aggregation and intermolecular disulfide formation. After elution from the resin the histidine tail can be removed using thrombin without cleaving the prion protein polypeptide chain. The same protocol as used here for hPrP has been successfully applied with bovine and murine prion proteins. The protein preparations are stable for weeks at room temperature in concentrated solution and are thus suitable for detailed structural studies. Preliminary biophysical characterization of hPrP(23-231) suggests that the C-terminal half of the polypeptide chain forms a well-structured globular domain, and that the N-terminal half does not form extensive regular secondary structures.
journal_name
FEBS Lettjournal_title
FEBS lettersauthors
Zahn R,von Schroetter C,Wüthrich Kdoi
10.1016/s0014-5793(97)01330-6subject
Has Abstractpub_date
1997-11-17 00:00:00pages
400-4issue
3eissn
0014-5793issn
1873-3468pii
S0014-5793(97)01330-6journal_volume
417pub_type
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