Premium
Human prion proteins expressed in Escherichia coli and purified by high‐affinity column refolding
Author(s) -
Zahn Ralph,
von Schroetter Christine,
Wüthrich Kurt
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)01330-6
Subject(s) - escherichia coli , histidine , inclusion bodies , chemistry , biochemistry , cytoplasm , agarose , affinity chromatography , prion proteins , amino acid , prion protein , gene , medicine , disease , pathology , enzyme
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 ( h PrP) 1 . 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 h PrP 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 h PrP(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.