GFABS: GFP-based biosensors possessing the binding properties of antibodies

Abstract

A family of GFP scaffolds capable of accommodating two proximal, randomized binding loops is disclosed. GFP-based binders binding with nanomolar affinity are developed from a library of these GFP scaffolds.

1 . A fluorescent biosensor comprising a fluorescent protein scaffold with heterologous amino acid sequence insertions between Glu172 and Asp173 and between Asp 102 and Asp 103, wherein the scaffold has the mutations D 19N, F64L, and A87T and wherein the protein is selected from the group of GFP and GFP variants. 
     
     
         2 . The biosensor of  claim 1  wherein the protein scaffold is green fluorescent protein. 
     
     
         3 . The biosensor of  claim 1  additionally comprising the mutation V163A. 
     
     
         4 . The biosensor of  claim 3  additionally comprising the mutations Y39H, N105T, D117G, E172K and L221V. 
     
     
         5 . The biosensor of  claim 4  additionally comprising the mutation F223S. 
     
     
         6 . The biosensor of  claim 1  wherein the fluorescence per molecule of the biosensor is at least 10% that of a non-loop inserted GFP. 
     
     
         7 . The biosensor of  claim 1  wherein the fluorescence per molecule of the biosensor is at least 20% that of a non-loop inserted GFP. 
     
     
         8 . The biosensor of  claim 1  wherein the fluorescence per molecule of the biosensor is at least 40% that of a non-loop inserted GFP. 
     
     
         9 . The biosensor of  claim 3  additionally comprising the mutation Y39H, wherein the heterologous amino acid sequence insertion at Glu172-Asp173 is selected from the group consisting of SEQ ID NO: 30 and SEQ ID NO: 34, and heterologous amino acid sequence insertion at Asp102-Asp103 is selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 33, wherein the biosensor is capable of specifically binding TrkB. 
     
     
         10 . The biosensor of  claim 4 , wherein the heterologous amino acid sequence insertion at Glu172-Asp173 is according to SEQ ID NO: 32, and heterologous amino acid sequence insertion at Asp102-Asp103 is according to SEQ ID NO: 31, wherein the biosensor is capable of specifically binding TrkB. 
     
     
         11 . The biosensor of  claim 4 , wherein the heterologous amino acid sequence insertion at Glu172-Asp173 is selected from the group consisting of SEQ ID NOs: 36, 38, 40 and 42, and heterologous amino acid sequence insertion at Asp102-Asp103 is selected from the group consisting of SEQ ID NOs: 35, 37, 39 and 41, wherein the biosensor is capable of specifically binding GAPDH. 
     
     
         12 . An expression library comprising multiple biosensors of  claim 1 , wherein the heterologous amino acid sequence insertions are not identical. 
     
     
         13 . An expression library comprising multiple biosensors of  claim 3 , wherein the heterologous amino acid sequence insertions are not identical. 
     
     
         14 . An expression library comprising multiple biosensors of  claim 4 , wherein the heterologous amino acid sequence insertions are not identical. 
     
     
         15 . An expression library comprising at least two fluorescent protein scaffolds, wherein the fluorescent protein scaffolds are not identical. 
     
     
         16 . A method of detecting an antigen comprising exposing the biosensor of  claim 1  to a specific antigen and detecting binding via fluorescence. 
     
     
         17 . A method of detecting an antigen comprising exposing the biosensor of  claim 3  to a specific antigen and detecting binding via fluorescence. 
     
     
         18 . A method of detecting an antigen comprising exposing the biosensor of  claim 4  to a specific antigen and detecting binding via fluorescence. 
     
     
         19 . A method of isolating a peptide sequence that will bind to a target molecule comprising exposing the target molecule to the library of  claim 12  and determining which library members bind to the target molecule. 
     
     
         20 . A fluorescent biosensor comprising a fluorescent protein scaffold with heterologous amino acid sequence insertions, wherein the scaffold has mutations corresponding to D19N, F64L and A87T, wherein the protein is selected from the group consisting of GFP and GFP variants. 
     
     
         21 . The fluorescent biosensor of  claim 20 , wherein the heterologous amino acid sequence insertions are located within any of the ten solvent-accessible loops. 
     
     
         22 . An expression library comprising the fluorescent biosensors of  claim 20 , wherein the heterologous amino acid sequence insertions are not identical.