Cell free translation system for compound screening and related uses

Abstract

The invention provides a cell-free system comprising not more than about 5% wheat germ extract for expressing proteins such as viral proteins and proteins required for viral capsid assembly, and proteins that assemble into multiprotein complexes in a manner analogous to viral capsids, are provided. Further provided are methods for expressing proteins such as viral proteins, proteins required for capsid assembly, and proteins that assemble into multiprotein complexes in a manner analogous to viral capsids using a cell-free system comprising not more than about 5% wheat germ extract. Further provided are methods to assay for compounds that modulate viral protein, viral capsid assembly, and assembly of proteins into multiprotein complexes whose disruption can ameliorate bacterial, parasitic, metabolic, oncologic, immunologic, or CNS disease in a cell-free system comprising not more than about 5% wheat germ extract.

1 . A cell-free system for expressing a protein of interest, wherein the cell-free system comprises not more than about 5% wheat germ extract and further comprises components necessary for expression of the protein of interest 
     
     
         2 . The cell-free system of  claim 1 , wherein the protein of interest is a viral protein. 
     
     
         3 . The cell-free system of  claim 2 , wherein the viral protein is a viral capsid protein. 
     
     
         4 . The cell-free system of  claim 1 , wherein the protein of interest is a capsid interacting protein. 
     
     
         5 . The cell free system of  claim 2 , wherein the viral protein is expressed by a virus which is a member of a viral family selected from the group consisting of Flaviviridae, Togaviridae, Bunyaviridae, Arenaviridae, Filoviridae, Poxviridae, Orthomyxoviridae, Rhabdoviridae, Herpesviridae, Coronaviridae, Paramyxoviridae, Hepadnaviridae, Bornaviridae, Picornaviridae, Retroviridae, Reoviridae, Papillomaviridae, Adenoviridae, Astroviridae, Polyomaviridae, Anelloviridae, Parvoviridae, Caliciviridae. 
     
     
         6 . The cell-free system of  claim 1 , wherein the components comprise a member selected from a buffer, an amino acid, a nucleic acid transcript and a combination thereof. 
     
     
         7 . The cell-free system of  claim 6 , wherein the components further comprise a member selected from a a detectable moiety, ATP, GTP, creatine phosphate, a labeled amino acid, myristoyl CoA lithium salt, an RNase inhibitor, creatine kinase, a tRNA and a combination thereof. 
     
     
         8 . The cell-free system of  claim 7 , wherein the labeled amino acid is [35S] methionine. 
     
     
         9 . The cell-free system of  claim 6 , wherein the nucleic acid transcript is assembled in an in vitro transcription reaction. 
     
     
         10 . The cell-free system of  claim 6 , wherein the nucleic acid transcript encodes a viral protein. 
     
     
         11 . The cell-free system of  claim 10 , wherein the nucleic acid transcript encodes a viral capsid protein. 
     
     
         12 . The cell-free system of  claim 6 , wherein the nucleic acid transcript encodes a viral capsid interacting protein. 
     
     
         13 . The cell-free system of  claim 6 , wherein the buffer comprises a member selected from the group consisting of potassium acetate, spermine, dithiothreitol and a combination thereof. 
     
     
         14 . A cell-free system for assembling a viral capsid, wherein the cell-free system comprises not more than about 5% wheat germ extract and components necessary for expression of at least one viral proteins comprising the viral capsid. 
     
     
         15 . The cell-free system of  claim 14 , wherein the viral capsid is from a viral family selected from Flaviviridae, Togaviridae, Bunyaviridae, Arenaviridae, Filoviridae, Poxviridae, Orthomyxoviridae, Rhabdoviridae, Herpesviridae, Coronaviridae, Paramyxoviridae, Hepadnaviridae, Bornaviridae, Picornaviridae, Retroviridae, Reoviridae, Papillomaviridae, Adenoviridae, Astroviridae, Polyomaviridae, Anelloviridae, Parvoviridae, Caliciviridae. 
     
     
         16 . The cell-free system of  claim 14 , wherein the components comprise a buffer, an amino acid, and a nucleic acid transcript encoding the viral protein. 
     
     
         17 . The cell-free system of  claim 16 , wherein the components further comprise a detectable moiety, ATP, GTP, creatine phosphate, a labeled amino acid, myristoyl CoA lithium salt, an RNase inhibitor, creatine kinase, and a tRNA. 
     
     
         18 . The cell-free system of  claim 17 , wherein the labeled amino acid is [35S] methionine. 
     
     
         19 . The cell-free system of  claim 16 , wherein the nucleic acid transcript is assembled in an in vitro transcription reaction. 
     
     
         20 . The cell-free system of  claim 16 , wherein the nucleic acid transcripts encode proteins that comprise the viral capsid. 
     
     
         21 . The cell-free system of  claim 16 , wherein the buffer comprises a member selected from the group consisting of potassium acetate, spermine, and dithiothreitol. 
     
     
         22 . A method of expressing a protein of interest, wherein the protein of interest is expressed using the cell-free system of  claim 1 . 
     
     
         23 . The method of  claim 22 , wherein the protein of interest is a viral protein. 
     
     
         24 . The method of  claim 23 , wherein the viral protein is a viral capsid protein. 
     
     
         25 . The method of  claim 22 , wherein the protein of interest is a viral capsid interacting protein. 
     
     
         26 . A method of assembling a viral capsid using the cell-free system of  claim 14 . 
     
     
         27 . A method of testing whether a compound modulates a cellular function, the method comprising
 (a) introducing the compound to the cell-free system of  claim 1 ; and   (b) determining whether the cellular function is modulated.   
     
     
         28 . The method of  claim 27 , wherein the compound is selected from the group consisting of a small organic molecule and a biopharmaceutical. 
     
     
         29 . A method of testing whether a compound modulates viral capsid assembly, the method comprising
 (a) introducing the compound to the cell-free system of  claim 14 ; and   (b) determining whether the viral capsid assembly is modulated.   
     
     
         30 . The method of  claim 29 , wherein the compound is selected from the group consisting of a small organic molecule and a biopharmaceutical. 
     
     
         31 . A kit for cell-free expression of a protein comprising:
 (a) a cell-free mixture comprising not more than about 5% wheat germ extract;   (b) components necessary for expression of the protein; and   (c) instructions sufficient for use of the kit.   
     
     
         32 . A kit for the assembly of a viral capsid comprising:
 (a) a cell-free mixture comprising not more than about 5% wheat germ extract;   (b) components necessary for expression of proteins required for viral capsid assembly; and   (c) instructions sufficient for use of the kit.   
     
     
         33 . A kit for determining whether a compound modulates expression of a protein comprising:
 (a) a cell-free mixture comprising not more than about 5% wheat germ extract;   (b) components necessary for expression of the protein; and   (c) instructions sufficient for determining whether the compound modulates expression of the protein.   
     
     
         34 . A kit for determining whether a compound modulates viral capsid assembly comprising:
 (a) a cell-free mixture comprising not more than about 5% wheat germ extract;   (b) components necessary for expression of proteins required for viral capsid assembly; and   (c) instructions sufficient for determining whether the compound modulates viral capsid assembly.   
     
     
         35 . A kit for determining whether a compound modulates multiprotein assembly comprising:
 (a) a cell-free mixture comprising not more than about 5% cellular extract;   (b) components necessary for expression of non-viral proteins; and   (c) instructions sufficient for determining whether the compound modulates multiprotein assembly.   
     
     
         36 . The cell-free system of  claim 1 , wherein the protein of interest is a non-viral protein that forms a multiprotein complex. 
     
     
         37 . The cell-free system of  claim 36 , wherein the multiprotein complex participates in diseases comprising central nervous system disorders, metabolic disorders, oncologic disorders, or immunologic disorders. 
     
     
         38 . The cell-free system of  claim 36 , wherein the protein of interest is a bacterial or parasitic protein. 
     
     
         39 . The method of  claim 26 , wherein the protein of interest is a non-viral protein that forms a multiprotein complex. 
     
     
         40 . The method of  claim 39 , wherein the multiprotein complex participates in diseases comprising central nervous system disorders, metabolic disorders, oncologic disorders, or immunologic disorders. 
     
     
         41 . The method of  claim 39 , wherein the protein of interest is a bacterial or parasitic protein.