Methods, compositions, and kits for protein crystallization

Abstract

The present invention provides methods, compositions, and kits for protein crystallization. The present invention involves electrophoretically focusing at least a first protein species within a matrix comprising at least 2 regions of different pH, the protein being present in amount sufficient to permit crystallization within said pH gradient.

1 . A method of crystallizing at least one protein, the method comprising: 
 electrophoretically focusing at least a first protein species within a matrix comprising at least 2 regions of different pH, the protein being present in amount sufficient to permit crystallization within said pH gradient.    
     
     
         2 . The method of  claim 1 , wherein said focusing is performed in a matrix having an immobilized continuous pH gradient.  
     
     
         3 . The method of  claim 2 , wherein said matrix is a polymer matrix.  
     
     
         4 . The method of  claim 3 , wherein said polymer matrix comprises cross-linked acrylamide monomers.  
     
     
         5 . The method of  claim 4 , wherein said continuous pH gradient is created by the covalent linkage within said polymer matrix of a plurality of species of buffering moieties having disparate pKa values.  
     
     
         6 . The method of  claim 5 , wherein said plurality of species of covalently linked buffering moieties are a plurality of acrylamido monomer species having disparate pKa values.  
     
     
         7 . The method of  claim 2 , wherein said polymer matrix is adherent to at least one solid backing member.  
     
     
         8 . The method of  claim 1 , wherein said at. least first protein species is initially present in inhomogeneous admixture with at least one additional protein species having a different isoelectric point.  
     
     
         9 . The method of  claim 8 , wherein each of said protein species is concurrently crystallized within said matrix at its respective isoelectric focal point.  
     
     
         10 . The method of  claim 1 , wherein said at least first protein species is a recombinantly expressed protein.  
     
     
         11 . The method of  claim 10 , wherein said recombinant expression is effected within a cell-free lysate.  
     
     
         12 . The method of  claim 1 , wherein said at least first protein species is electrophoretically focused in the substantial absence of detergent.  
     
     
         13 . The method of  claim 7 , wherein said at least one solid backing member and the polymer matrix adhered thereto is fashioned in the shape of a strip, wherein said strip is a prior-cast, dehydrated, immobilized pH gradient (IPG) strip.  
     
     
         14 . The method of  claim 13 , wherein electrophoretic focusing of said at least first protein species comprises: 
 hydratingly lodging the prior-cast, dehydrated, IPG strip within an enclosing member that permits spaced electrical communication with said strip; and then using said spaced electrical communication to establish a voltage gradient in the polymer matrix of said strip sufficient to effect electrophoretic focusing of proteins therein.    
     
     
         15 . The method of  claim 14 , further comprising the prior step of inserting said prior-cast IPG strip in its dehydrated state within said enclosing member.  
     
     
         16 . The method of  claim 14 , wherein said step of hydratingly lodging comprises: 
 contacting said enclosed dehydrated IPG strip with an aqueous solution for a time sufficient to lodge said separation medium within said enclosing member.    
     
     
         17 . The method of  claim 14 , wherein said IPG strip isoelectric focusing is performed in a system comprising: 
 means for enclosing a plurality of said strips, said enclosing means permitting spaced electrical communication separately with each of said enclosed strips through respective first and second entries; and    means responsive to an external compressive force for effecting spaced electrical communication by a single anode and single cathode simultaneously with each of said enclosed strips, wherein said electrical communication means is capable of distributing an external compressive force to urge said anode and said cathode toward said enclosing means with greater pressure at said first and second entries than elsewhere on said enclosing means.    
     
     
         18 . The method of  claim 1 , further comprising the later step of: 
 isolating crystals of said at least first protein species.    
     
     
         19 . The method of  claim 18 , wherein said at least 2 regions of different pH are immobilized within said matrix, and said isolating step comprises separately excising the portion of said matrix in which said crystals of the at least first protein species are embedded therein.  
     
     
         20 . The method of  claim 1 , further comprising the step, after electrophoretically focusing, of: 
 using said crystallized first protein species to nucleate further crystallization of said respective protein species from solution.    
     
     
         21 . The method of  claim 1 , further comprising the step of: 
 analyzing said at least first protein species.    
     
     
         22 . The method of  claim 21 , wherein said analysis step comprises diffraction analysis.  
     
     
         23 . The method of  claim 22 , wherein said diffraction analysis comprises X-ray diffraction analysis.  
     
     
         24 . The method of  claim 21 , wherein said analysis step comprises determining at least a partial 3D structure of said protein species.  
     
     
         25 . The method of  claim 1 , wherein said at least first protein species is first complexed with a compound chosen from a chemical library.  
     
     
         26 . A composition, comprising: 
 at least one crystal of at least a first protein species; and    a polymer matrix,    wherein the polymer matrix comprises a plurality of species of pH-conferring moieties, each of the plurality having a distinct pKa.    
     
     
         27 . The composition of  claim 26 , wherein at least one of said at least one crystal of said at least first protein species is a crystal of the protein in a native, non-denatured, conformation.  
     
     
         28 . The composition of  claim 26 , wherein said polymer matrix is an acrylamide polymer.  
     
     
         29 . The composition of  claim 26 , wherein said polymer matrix is adherent to at least one support backing.  
     
     
         30 . A kit, comprising: 
 a polymer matrix having a continuous pH gradient; and at least one of:    instructions for performing the method of  claim 1 , an x-ray diffraction holder, and a protein crystal transport tool.    
     
     
         31 . The kit of  claim 30 , wherein said polymer matrix is part of an IPG strip.  
     
     
         32 . The kit of  claim 30 , wherein the kit comprises a protein crystal transport tool, whereby the tool is effective for excising a protein crystal from said polymer matrix and transporting the protein crystal to an x-ray diffraction holder.  
     
     
         33 . The kit of  claim 30 , wherein the kit comprises an x-ray diffraction holder.  
     
     
         34 . The kit of  claim 30 , wherein the kit comprises one or more protein crystal transport tools, which together are effective for excising a portion of the polymer matrix comprising the protein crystals and effective for transporting the portion of the polymer matrix comprising the protein crystals to an x-ray diffraction holder.  
     
     
         35 . A method of crystallizing at least one protein, comprising electrophoretically transporting at least a first protein species within a polymer matrix that includes at least 2 regions of different pH, wherein 
 the isoelectric point of the protein falls outside of the pH regions of the polymer matrix, and    the protein being present in an amount sufficient to permit formation of protein crystals at a terminal region of the polymer matrix.    
     
     
         36 . The method of  claim 35 , wherein the polymer matrix comprises a continuous pH gradient.  
     
     
         37 . The method of  claim 35 , further comprising excising the terminal region of the polymer matrix that includes the protein crystals.  
     
     
         38 . The method of  claim 37 , further comprising analyzing the protein crystals using x-ray diffraction.  
     
     
         39 . The method of  claim 37 , wherein the excision step is performed using a protein crystal transport tool.  
     
     
         40 . The method of  claim 39 , further comprising transporting=the excised terminal region of the polymer matrix to an x-ray diffraction holder.