Functional protein arrays

Abstract

The present invention relates to a method for producing a protein array starting from DNA (or mRNA) in which a number of native, functional proteins, domains or peptides are produced in parallel by in in vitro synthesis using a cell free system for transcription and translation. The products are immobilised in a gridded format on a surface, using an isolation sequence tag incorporated into the proteins.

1 - 35 . (canceled) 
     
     
         36 . A method for creating a protein array, wherein:
 (a) DNA constructs are prepared containing sequences allowing transcription and translation by a cell free system and sequences enabling covalent or noncovalent attachment of the encoded protein, domain or peptide to a surface or bead;   (b) the DNA constructs are distributed in a gridded format into wells, onto surfaces or in the presence of a bead, any of which carries a ligand or reagent for immobilization of the proteins;   (c) the DNA is transcribed and translated, in the gridded format, using a cell free system; and   (d) the proteins, domains or peptides produced become attached to the well, surface or bead as they are produced to create the protein array.   
     
     
         37 . A method for creating a protein array, wherein:
 (a) DNA constructs are prepared containing sequences allowing transcription and translation by a cell free system and sequences enabling covalent or noncovalent attachment of the encoded protein, domain or peptide to a surface or bead;   (b) the DNA is distributed in a gridded format and immobilized either covalently or noncovalently, in wells, on a surface or a bead, any of which carry a ligand or reagent for immobilization of the proteins;   (c) the DNA is transcribed and translated, in the gridded format, using a cell free system; and   (d) the proteins, domains or peptides produced become attached to the well, the surface or bead as they are produced, to create the protein array.   
     
     
         38 . The method according to  claim 36 , wherein the DNA is first transcribed into mRNA and the mRNA then translated in a separate reaction. 
     
     
         39 . The method according to  claim 36 , wherein the cell free system enables coupled transcription and translation of DNA, or allows separate transcription of DNA and translation of mRNA, and is of rabbit reticulocyte, wheat germ, yeast, bacterial or other eukaryotic or prokaryotic origin. 
     
     
         40 . A protein array created using a cell free in vitro system for transcription and translation of DNA molecules or translation of mRNA molecules, wherein the DNA or mRNA molecules are distributed in a gridded format, to produce individual proteins, domains or peptides, which contain amino acid sequences enabling covalent or noncovalent attachment to a surface or bead, wherein translation takes place on a surface or in the presence of a bead carrying an appropriate ligand or reagent for immobilization of the proteins, such that individual proteins become attached to the surface or bead as they are produced, in which the proteins, domains or peptides include double hexahistidine sequences for immobilization through interaction with metal ions such as nickel and in which the two hexahistidine sequences of the double hexahistidine sequence are separated by an amino acid spacer sequence. 
     
     
         41 . The protein array according to  claim 40 , in which the amino acid spacer sequence is about 11 amino acids in length. 
     
     
         42 . The protein array according to  claim 40 , in which the double hexahistidine sequences are separated by a peptide spacer containing (his)6-SRAWRHPQFGG-(his)6 (SEQ ID NO: 2). 
     
     
         43 . The protein array according to  claim 40 , in which the DNA or mRNA molecules are covalently or noncovalently bound to a surface or bead, and wherein the surface or bead also carries a ligand or reagent for immobilization of the proteins, such that the individual proteins become attached to the surface or bead as they are produced. 
     
     
         44 . The protein array according to  claim 40 , which is created from DNA constructs, which can be expressed in prokaryotic or eukaryotic cell free systems as a result of inclusion of appropriate primer sequences. 
     
     
         45 . The protein array according to  claim 40 , which is created using cell free systems, which enable coupled transcription and translation of DNA, or which allow separate transcription of DNA and translation of mRNA, and are of rabbit reticulocyte, wheat germ, yeast, bacterial or other eukaryotic or prokaryotic origin. 
     
     
         46 . The protein array according to  claim 40 , in which coupled transcription or translation of DNA, or separate translation of mRNA, is carried out in microwells or on the surface of glass, nitrocellulose or other membrane, or other supporting medium or in the presence of beads. 
     
     
         47 . The protein array according to  claim 40 , in which the proteins arrayed are single chain antibody fragments. 
     
     
         48 . The protein array according to  claim 40 , in which the proteins arrayed retain and display their native function. 
     
     
         49 . A method of identifying interactions of an arrayed protein with other molecules using a protein array of  claim 40 , wherein the other molecules include antibodies, proteins or domains, peptides, small ligands, cell extracts, and nucleic acids. 
     
     
         50 . A method of identifying interactions of an arrayed protein with other molecules using a protein array of  claim 40 , wherein the other molecules include molecules displayed in a library, such as a phage display or ribosome display library. 
     
     
         51 . A method of studying cellular protein expression profiles comprising a protein array of  claim 40 . 
     
     
         52 . A method of studying post-translational modifications of cellular proteins comprising a protein array of  claim 40 .