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Illegal Beings: Human Clones
Human prion proteins expressed in Escherichia coli and purified by high-affinity column refo
Zahn R, von Schroetter C, Wuthrich KFEBS Lett. 1997 Nov 17;417(3):400-4..
Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische Hochschule Honggerberg, Zurich, Switzerland.
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.
This Message is being posted for educational purposes, as well as for comment and criticism, by the visitors to the HumanCloning.org Foundation website (http://www.humancloning.org ).
Disclaimer: This abstract is being posted for educational purposes, as well as for comment and criticism, by the visitors to the Human Cloning Foundation website (www.HumanCloning.org ). This abstract is representative of a larger article that is indexed on Medline.
The Human Cloning Foundation was established February, 1988. .
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