Compositions for detecting ras gene proteins and cancer therapeutics

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.

What is claimed is: 
     
       1. 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. 
     
     
       2. 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.   
     
     
       3. The method as described in claim 1 wherein said molecule is purified from human placenta. 
     
     
       4. The method as described in claim 1 wherein said purification is conducted in solutions containing one or more protease inhibitors at concentrations that effectively inhibit proteolysis of said molecule. 
     
     
       5. The method as described in claim 4 wherein said molecule is present in a solution comprising a reducing agent in an amount effective to preserve the activity of said molecule. 
     
     
       6. The method as described in claim 5 wherein said purification is conducted in solutions containing one or more metal ion chelators at concentrations that prevent substantial loss of activity of said molecule. 
     
     
       7. The method as described in claim 6 wherein said purification is conducted in solutions containing cations in an amount that substantially maintains the activity of said molecule. 
     
     
       8. The method as described in claim 7 wherein said cation is magnesium chloride. 
     
     
       9. The method as described in claim 1 wherein said solution contains about 1 mM magnesium chloride, 5 mM EGTA, 0.1 mM DTT, and 100 μM PMSF. 
     
     
       10. The method as described in claim 1 wherein said tirst cation exchange chromatographic material is S-Sepharose. 
     
     
       11. The method as described in claim 1 wherein said anion exchange chromatographic material is diethyl aminoethyl. 
     
     
       12. The method as described in claim 1 wherein said second cation chromatographic material comprises sulfopropyl functional groups. 
     
     
       13. The method as described in claim 10 wherein said second cation exchange material is TSK-SE-5-PW. 
     
     
       14. The method as described in claim 1 wherein a hydrophobic interaction chromatographic material is substituted for said second cation exchange material, and said second eluate from said anion exchange chromatographic material is made compatible with said hydrophobic interaction chromatographic material by increasing the ionic strength of said second eluate, and forming a third eluate by eluting said molecule from said hydrophobic interaction chromatographic material. 
     
     
       15. The method as described in claim 14 wherein said molecule is eluted from said hydrophobic interaction chromatographic material with an aqueous low salt solution, or an effective mount of a polyol. 
     
     
       16. The method as described in claim 14 wherein said hydrophobic interaction chromatographic material is under high pressure. 
     
     
       17. An isolated and substantially pure protein molecule as described in claim 1, wherein said non-oncogenie ras p21 GTPase activity is associated with H-ras. 
     
     
       18. An isolated and substantially pure protein molecule as described in claim 1, wherein said non-oncogenic ras p21 GTPase activity is associated with N-ras.