Versatile display scaffolds for proteins

Abstract

Provided are processes and materials for solving biological or structural information about proteins or other organic molecules. The processes capitalize on a rigid multimeric nanocage formed from self-assembling substructure proteins. The processes and materials allow for recognition and tight, optionally covalent, bonding of any protein molecule with a tag complementary to a capture sequence on the nanocage. The processes and materials may be used to obtain biological or structural information by cryo-electron microscopy and overcome prior limitations of target protein size or salt concentration.

1 .- 27 . (canceled) 
     
     
         28 . A process of immunizing a subject, comprising administering to the subject a three dimensional protein structure comprising:
 a multimeric self-assembling nanocage, the nanocage comprising a plurality of protein substructures, one or more of the protein substructures comprising a linker and a capture sequence on an N-terminus; and   a target protein, the target protein comprising a tag, the tag complementary to the capture sequence to that an association between the tag and the capture sequence covalently bonds said target protein to said protein substructure, or non-covalently bonds said target protein to said protein substructure with a K D  or 10 12  M or lower.   
     
     
         29 . The process of  claim 28 , wherein said linker is intermediate the protein substructure and the capture sequence, the linker covalently bonding the capture sequence to the protein substructure. 
     
     
         30 . The process of  claim 29 , wherein the linker is a flexible linker. 
     
     
         31 . The process of  claim 30 , wherein the flexible linker is selected from the group consisting of a multimer of the amino acid sequence GGS, GSS, or combinations thereof. 
     
     
         32 . The process of  claim 29 , wherein the linker is a rigid linker. 
     
     
         33 . The process of  claim 32 , wherein the rigid linker comprises one or more stabilizing disulfide bonds, one or more repeats of SEQ ID NO: 10, an amino acid sequence comprising 3 or more proline residues, an amino acid sequence comprising 1 or more sequences of PPA, or a combination thereof. 
     
     
         34 . The process of  claim 28 , wherein each of said protein substructures comprises an amino acid sequence that is 70% or greater identical to any one of SEQ ID NOs:1-6. 
     
     
         35 . The process of  claim 28 , wherein the capture sequence comprises the sequence of SEQ ID NO: 8, SEQ ID NO: 9, biotin, or avidin. 
     
     
         36 . The process of  claim 28 , wherein the multimeric self-assembling protein structure comprises a multimer of any one of SEQ ID NOs: 1-6. 
     
     
         37 . The process of  claim 28 , wherein the multimer is a 60-mer. 
     
     
         38 . The process of  claim 28 , wherein the multimeric self-assembling protein structure forms a dodecahedron. 
     
     
         39 . The process of  claim 28 , wherein the target protein has a molecular weight of less than 200 kDa. 
     
     
         40 . The process of  claim 28 , wherein the target protein has a molecular weight of less than 150 kDa. 
     
     
         41 . The process of  claim 28 , wherein the target protein has a molecular weight of less than 120 kDa. 
     
     
         42 . The process of  claim 28 , wherein the tag comprises SEQ ID NO: 20, SEQ ID NO: 21, biotin, or avidin. 
     
     
         43 . The process of  claim 28 , wherein said target protein is the RNA-binding protein, cytosolic Poly-A Binding Protein (PABP), a DNA-binding protein of the ApiAP2 specific transcription factor family, a binding domain of tristetraprolin (TTP) of a NOT family protein, or a RNA-recognition motif of the Upregulated in Infectious Sporozoites 12 (UIS12) protein. 
     
     
         44 . The process of  claim 28 , wherein said target protein is saturated onto said multimeric self-assembling protein structure to form a target complex at a level of 50% or greater. 
     
     
         45 . The process of  claim 28 , wherein said target protein is saturated onto said multimeric self-assembling protein structure to form a target complex at a level of 90% or greater. 
     
     
         46 . The process of  claim 28 , wherein the protein structure is in an aqueous buffer comprising at or greater than 100 mM of a salt. 
     
     
         47 . The process of  claim 46 , wherein the salt is 200 mM to 500 mM.