US2005233428A1PendingUtilityA1

Method of affinity purifying proteins using modified bis-arsenical fluorescein

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Assignee: UNIV CALIFORNIAPriority: Jan 24, 2000Filed: Dec 14, 2004Published: Oct 20, 2005
Est. expiryJan 24, 2020(expired)· nominal 20-yr term from priority
Inventors:Ronald D. ValeKurt S. ThornRoger CookeMarija MatuskaNariman Naber
C07F 9/80C07K 1/22C07K 19/00
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Claims

Abstract

The present invention features methods for purifying polypeptides of interest using a modified Fluorescein arsenical helix binder (FlAsH) compound immobilized on a solid support. An exemplary FlAsH target sequence motif is also presented. Examples of modification of the FlAsH compound which allow immobilization to a solid support are also provided. The present invention also provides DNA constructs for producing a dual affinity tagged polypeptide and methods for purification thereof.

Claims

exact text as granted — not AI-modified
1 - 37 . (canceled)  
     
     
         38 . A method for isolating a polypeptide of interest, which has at its N-terminus a genetically encoded affinity tag and at its C-terminus a FlAsH sequence motif comprising; 
 a) contacting a solution which contains a polypeptide of interest with an affinity resin which binds to the affinity tag;    b) eluting the polypeptides bound to the affinity column;    c) contacting the modified FlAsH compound immobilized on a solid support with the polypeptides from step b), under conditions that allow binding of the polypeptide to the FlAsH compound; and    d) eluting the polypeptide of interest from the immobilized FlAsH compound.    
     
     
         39 . The method of  claim 38 , wherein the affinity tag is selected from the group consisting of polyhistidine, maltose binding protein, glutathione S-transferase, and the FLAG tag.  
     
     
         40 . The method of  claim 39 , wherein the polyhistidine tag is the 6×histidine (His6) tag.  
     
     
         41 . The method of  claim 38 , wherein the FlAsH compound is immobilized to a solid support selected from the group consisting of agarose, polyacrylimide, glass, ceramics, natural or synthetic polymeric materials, beads, coverslips, paper, metals, metalloids, polacryloylmorpholide, various plastics and plastic copolymers such as Nylon™, Teflon™, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polystyrene, polystyrene/latex, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and controlled-pore glass, aerogels, and affinity exchange resins.  
     
     
         42 . The method of  claim 38 , wherein the polypeptide of interest is eluted from the affinity resin with a solution of imadizole.  
     
     
         43 . The method of  claim 38 , wherein the polypeptide of interest is eluted from the affinity resin with a low pH solution.  
     
     
         44 . The method of  claim 38  wherein the polypeptide of interest is eluted from the FlAsH compound using a dithiol solution.  
     
     
         45 . The method of  claim 44  wherein said dithiol solution is selected from the group consisting of 1,2 Ethanedithiol (EDT), Dithiotheritol (DTT), 2,3 and Dimercaptopropanesulfonate (DMPS).  
     
     
         46 . A method for isolating a polypeptide of interest, which has at its N-terminus a genetically-encoded affinity tag and at its C-terminus a FlAsH target sequence motif comprising: 
 a) contacting the solution which contains a polypeptide of interest with a FlAsH compound immobilized to a solid support;    b) eluting the polypeptides bound to the immobilized FlAsH compound;    c) contacting an affinity resin with the polypeptide solution from step b, under conditions that allow binding of the polypeptide to the affinity resin; and    d) eluting the polypeptide of interest from the affinity resin.    
     
     
         47 . The method of  claim 46  wherein the polypeptide of interest has been modified by the addition of the FlAsH target sequence motif C C X 1  X 2  C C where X 1  and X 2  are any amino acid.  
     
     
         48 . The method of  claim 47  wherein X 1  and X 2  are the same amino acid.  
     
     
         49 . The method of  claim 47  wherein X 1  and X 2  are different amino acids.  
     
     
         50 . The method of  claim 46  wherein the FlAsH compound is immobilized to a solid support selected from the group consisting of agarose, polyacrylimide, glass, ceramics, natural or synthetic polymeric materials, beads, coverslips, paper, metals, metalloids, polacryloylmorpholide, various plastics and plastic copolymers such as Nylon™, Teflon™, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polystyrene, polystyrene/latex, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and controlled-pore glass, aerogels, and affinity exchange resins.  
     
     
         51 . The method of  claim 46  wherein the polypeptide of interest is eluted from the FlAsH compound using a dithiol solution.  
     
     
         52 . The method of  claim 51  wherein said dithiol solution is selected from the group consisting of 1,2 Ethanedithiol (EDT), Dithiotheritol (DTT), 2,3 and Dimercaptopropanesulfonate (DMPS).  
     
     
         53 . The method of  claim 46 , wherein the affinity tag is selected from the group consisting of polyhistidine, maltose binding protein, glutathione S-transferase, and the FLAG tag.  
     
     
         54 . The method of  claim 53 , wherein the polyhistidine tag is the 6×histidine (His6) tag.  
     
     
         55 . The method of  claim 46 , wherein the polypeptide of interest is eluted from the affinity resin with a solution of imadizole.  
     
     
         56 . The method of  claim 46 , wherein the polypeptide of interest is eluted from the affinity resin with a low pH solution.  
     
     
         57 - 76 . (canceled)  
     
     
         77 . A method for isolating a polypeptide of interest, which has at its N-terminus a FlAsH target sequence motif and at its C-terminus a genetically-encoded affinity tag comprising; 
 a) contacting a solution which contains a polypeptide of interest with an affinity resin which binds to the affinity tag;    b) eluting the polypeptides bound to the affinity column;    c) contacting the modified FlAsH compound immobilized on a solid support with the polypeptides from step b), under conditions that allow binding of the polypeptide to the FlAsH compound; and    d) eluting the polypeptide of interest from the immobilized FlAsH compound.    
     
     
         78 . The method of  claim 77 , wherein the affinity tag is selected from the group consisting of polyhistidine, maltose binding protein, glutathione S-transferase, and the FLAG tag.  
     
     
         79 . The method of  claim 78 , wherein the polyhistidine tag is the 6×histidine (His6) tag.  
     
     
         80 . The method of  claim 77  wherein the polypeptide of interest has been modified by the addition of the FlAsH target sequence motif C C X 1  X 2  C C where X 1  and X 2  are any amino acid.  
     
     
         81 . The method of  claim 77  wherein X 1  and X 2  are the same amino acid.  
     
     
         82 . The method of  claim 77  wherein X 1  and X 2  are different amino acids.  
     
     
         83 . The method of  claim 77 , wherein the FlAsH compound is immobilized to a solid support selected from the group consisting of agarose, polyacrylimide, glass, ceramics, natural or synthetic polymeric materials, beads, coverslips, paper, metals, metalloids, polacryloylmorpholide, various plastics and plastic copolymers such as Nylon™, Teflon™, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polystyrene, polystyrene/latex, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and controlled-pore glass, aerogels, and affinity exchange resins.  
     
     
         84 . The method of  claim 77 , wherein the polypeptide of interest is eluted from the affinity resin with a solution of imadizole.  
     
     
         85 . The method of  claim 77 , wherein the polypeptide of interest is eluted from the affinity resin with a low pH solution.  
     
     
         86 . The method of  claim 77  wherein the polypeptide of interest is eluted from the FlAsH compound using a dithiol solution.  
     
     
         87 . The method of  claim 86  wherein said dithiol solution is selected from the group consisting of 1,2 Ethanedithiol (EDT), Dithiotheritol (DTT), 2,3 and Dimercaptopropanesulfonate (DMPS).  
     
     
         88 . A method for isolating a polypeptide of interest, which has at its N-terminus a FlAsH target sequence motif and at its C-terminus a genetically-encoded affinity tag comprising; 
 a) contacting the solution which contains a polypeptide of interest with a FlAsH compound immobilized to a solid support;    b) eluting the polypeptides bound to the immobilized FlAsH compound;    c) contacting an affinity resin with the polypeptide solution from step b, under conditions that allow binding of the polypeptide to the affinity resin; and    d) eluting the polypeptide of interest from the affinity resin.    
     
     
         89 . The method of  claim 88  wherein the polypeptide of interest has been modified by the addition of the FlAsH target sequence motif C C X 1  X 2  C C where X 1  and X 2  are any amino acid.  
     
     
         90 . The method of  claim 88  wherein X 1  and X 2  are the same amino acid.  
     
     
         91 . The method of  claim 88  wherein X 1  and X 2  are different amino acids.  
     
     
         92 . The method of  claim 88  wherein the FlAsH compound is immobilized to a solid support selected from the group consisting of agarose, polyacrylimide, glass, ceramics, natural or synthetic polymeric materials, beads, coverslips, paper, metals, metalloids, polacryloylmorpholide, various plastics and plastic copolymers such as Nylon™, Teflon™, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polystyrene, polystyrene/latex, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and controlled-pore glass, aerogels, and affinity exchange resins.  
     
     
         93 . The method of  claim 88  wherein the polypeptide of interest is eluted from the FlAsH compound using a dithiol solution.  
     
     
         94 . The method of  claim 93  wherein said dithiol solution is selected from the group consisting of 1,2 Ethanedithiol (EDT), Dithiotheritol (DTT), 2,3 and Dimercaptopropanesulfonate (DMPS).  
     
     
         95 . The method of  claim 88 , wherein the affinity tag is selected from the group consisting of polyhistidine, maltose binding protein, glutathione S-transferase, and the FLAG tag.  
     
     
         96 . The method of  claim 95 , wherein the polyhistidine tag is the 6×histidine (His6) tag.  
     
     
         97 . The method of  claim 88 , wherein the polypeptide of interest is eluted from the affinity resin with a solution of imadizole.  
     
     
         98 . The method of  claim 88 , wherein the polypeptide of interest is eluted from the affinity resin with a low pH solution.  
     
     
         99 - 103 . (canceled)

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