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.

We claim: 
     
         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 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 at least one 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 transcript encodes 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 . The cell-free system of  claim 1 , wherein the protein of interest is a non-viral protein that forms a multiprotein complex. 
     
     
         23 . The cell-free system of  claim 22 , wherein the multiprotein complex participates in diseases comprising central nervous system disorders, metabolic disorders, oncologic disorders, or immunologic disorders. 
     
     
         24 . The cell-free system of  claim 22 , wherein the protein of interest is a bacterial or parasitic protein. 
     
     
         25 . 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.   
     
     
         26 . 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.