Systematic Creation of Fluorescent Fusion Polypeptides

Abstract

A method for creating a plasmid for use in producing a chimeric antibody, comprising (a) receiving a FAB region of the antibody; (b) receiving a fluorescent protein; (c) receiving a linker having length of at least 5 amino acids; (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.

We claim: 
     
         1 . A method of creating a plasmid for use in producing a chimeric antibody, comprising:
 (a) receiving a FAB region of the antibody;   (b) receiving a fluorescent protein;   (c) receiving a linker comprising at least four glycine residues and one serine residue;   (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.   
     
     
         2 . The method of  claim 1 , further comprising using PCR to produce volumes of the FAB region, the fluorescent protein, and the linker for use in the Gibson assembly process. 
     
     
         3 . The method of  claim 1 , wherein receiving a FAB region comprises determining if the combined length of the FAB region, the fluorescent protein, and the linker will exceed the Durvasula length, and, if so, modifying the FAB region such that the combined length will not exceed the Durvasula length. 
     
     
         4 . The method of  claim 1 , wherein receiving a linker comprises determining the amount the length of (a) the combined length of the FAB region and the fluorescent protein, is less than (b) the Durvasula length, and receiving a linker whose length equals the determined amount. 
     
     
         5 . The method of  claim 1 , further comprising determining the difference in length between (a) the combined length of the FAB region and the fluorescent protein, and (b) the Durvasula length, and,
 (x) if the difference is less than or equal to 5 amino acids, then modifying the FAB region by shortening the length of the FAB region by the amount the difference is less than 5 amino acids and using a linker having a length of 5 amino acids;   (y) if the difference greater than 5 amino acids, then using a linker having a length equal to the difference.   
     
     
         6 . A method of producing a chimeric antibody, comprising (a) creating a plasmid according to  claim 1 , (b) inserting the plasmid into at least one of (y) chemically competent bacteria capable of protein expression from the plasmid, (z) electrocompetent bacteria capable of protein expression from the plasmid, and (c) using the bacteria to produce the chimeric antibody. 
     
     
         7 . The method of  claim 6  wherein step (b) comprises inserting the plasmid into chemically competent bacteria capable of protein expression from the plasmid. 
     
     
         8 . The method of  claim 6  wherein step (b) comprises inserting the plasmid into electrocompetent bacteria capable of protein expression from the plasmid. 
     
     
         9 . The method of  claim 6  wherein the bacteria comprises  E. coli.    
     
     
         10 . The method of  claim 6 , wherein the plasmid is produced according to the method of  claim 5 . 
     
     
         11 . The method of  claim 1 , wherein the linker comprises at least one natural amino acid. 
     
     
         12 . The method of  claim 1 , wherein the linker comprises at least one synthetic amino acid. 
     
     
         13 . The method of  claim 1 , wherein the linker comprises at least one natural amino acid and at least one synthetic amino acid.