Compositions for detecting ras gene proteins and cancer therapeutics
AssigneeCetus Oncology Corporation
InventorsFrancis P. McCormick, Kirston E. Koths, Robert F. Halenbeck, Mary M. Trahey
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Abstract
Compositions useful for detecting ras gene proteins are described consisting of GTP and a protein having an apparent reduced molecular weight of about 115,000-120,000 daltons, or fragments derived therefrom, that stimulate ras protein guanosine triphosphatase activity. Also described are methods whereby the compositions are used to identify cancer therapeutics.
Claims18
A isolated and substantially pure protein molecule comprising art apparent molecular weight of about 115,000-120,000 daltons as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis run under reducing condition that simulate non-oncogenic ras p21 GTPase activity but does not substantially affect GTPase activity of oncogenic routants.
A method of purifying a molecule having art apparent reduced molecular weight of about 115,000-120,000 daltons that simulates non-oncogenic ras p21 GTPase activity, but does not substantially affect GTPase activity of oncogenic routants, from a solution containing the same, comprising the steps of:
- contacting said solution with cation exchange chromatographic material for a time sufficient for said molecule to bind to said material;
- forming a first eluate containing said molecule by eluting said molecule from said cation chromatographic material by contacting said chromatographic material with an aqueous salt solution;
- identifying fractions in said first eluate having said molecular, and reducing the salt concentration present in said fractions to be compatible with anion exchange chromatography;
- forming a second eluate by contacting said fractions of said first eluate containing said molecule with artion exchange chromatographic material for a time sufficient for said molecule to bind to said material, and eluting said molecule from said material artion exchange material with art aqueous salt solution;
- forming a third eluate by contacting said second eluate with a second cation exchange chromatographic material for a time sufficient for said molecule to bind to said material, and eluting said material from said second cation exchange chromatographic material;
- and
- identifying fractions of said third eluate containing said molecule.
Dependencies are inferred from the claim text.