Compositions for detecting ras gene proteins and cancer therapeutics
AssigneeCetus Oncology Corporation
InventorsFrank 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.
Claims8
An isolated and substantially pure protein fragment comprising an apparent molecular weight of about 45,000-55,000 daltons as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis run under reducing conditions, said fragment having the properties of stimulating non-oncogenic ras p21 GTPase activity, but does not substantially affect GTPase activity of oncogenic mutants.
A method for purifying a protein fragment having an apparent reduced molecular weight of about 45,000-55,000 daltons that stimulates non-oncogenic ras p21 GTPase activity, but does not substantially affect GTPase activity of oncogenic mutants from a solution containing the same, comprising the steps of:
- a) contacting said solution with cation exchange chromatographic material for a time sufficient for said molecule to bind to said material;
- b) 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;
- c) identifying fractions in said first eluate having said molecule, and reducing the salt concentration present in said fractions to be compatible with anion exchange chromatography;
- d) forming a second eluate by contacting said fractions of said first eluate containing said molecule with anion exchange chromatographic material for a time sufficient for said molecule to bind to said material, and eluting said molecule from said material anion exchange material with an aqueous salt solution;
- e) 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
- f) identifying fractions of said third eluate containing said molecule.
A method for screening potential cancer therapeutics, comprising the steps of:
- (a) combining in a GAP assay compatible solution a compound suspected of being a cancer therapeutic, ras p21 protein having GAP stimulatable GTPase activity, and labeled GTP;
- (b) measuring the amount of GTP converted to GDP plus phosphate;
- and
- (c) relating the amount of GTP converted to GDP with a control sample prepared in accordance with step "a", said control sample being known to be free of said suspected cancer therapeutic.
Dependencies are inferred from the claim text.