Combined ligand and receptor display

Abstract

A method for selecting nucleic acid sequences encoding ligand and receptor molecules capable of specific binding to each other is disclosed in which nucleic acid encoding ligand or receptor molecules is expressed in a host microorganism in combination with a surface molecule, such as E. coli pili, so that the ligand or receptor are displayed on the surface of the host microorganism. A replicable genetic unit, such as a filamentous bacteriophage, is used to display candidate binding partners to the ligand or receptor, with the binding of the ligand or receptor to the candidate binding partner mediating the transfer of nucleic acid from the replicable genetic unit to the microorganism. The method can be highly selective as the host microorganism is modified so that it does not display the surface molecule other than as a fusion with the ligand receptor molecule. The method is rapid and simple and opens up new applications based on the detection of ligand and receptors where both are unknown.

1 . A a method for selecting nucleic acid sequences encoding ligand and receptor molecules capable of specific binding to each other, the method comprising: 
 (a) expressing in a host microorganism nucleic acid encoding a surface molecule which is operably linked to the expression of nucleic acid encoding a ligand or receptor molecule, or a functional fragments thereof, so that the microorganism expresses the surface molecule and the ligand or receptor molecule as a fusion and displays it on its surface, wherein the host microorganism is modified so that it does not display the surface molecule other than as a fusion with the ligand or receptor molecule;    (b) contacting the modified host microorganism of step (a) with one or more replicable genetic units capable of expressing nucleic acid encoding ligand or receptor molecules, or functional fragments thereof, the ligand or receptor molecules being candidates for specific binding to the molecules displayed by the host microorganisms of step (a) and being expressed as fusions with a surface protein of the replicable genetic unit, wherein binding of the surface displayed ligand and receptor molecules mediates the transfer of the nucleic acid encoding the ligand or receptor from the replicable genetic unit to the microorganism; and,    (c) selecting the host microorganisms containing the nucleic acid sequences encoding the ligand and receptor molecules, or functional fragments thereof.    
     
     
         2 . A method for selecting specific ligand and receptor encoding sequences wherein: 
 (a) the expression of a surface molecule encoding sequence is combined with a ligand encoding sequence, or a sequence encoding a functional fragment thereof, so that the host microorganism expresses modified ligand molecules on its surface;    (b) infecting, or in other ways transferring DNA to, the modified host organism of step (a) with a genetically modified replicable genetic unit capable of expressing a receptor, or a functional fragment thereof, fused to a surface protein of the replicable genetic unit; and,    (c) selecting infected host organisms containing specific ligand and receptor encoding sequences or sequences encoding functional fragments thereof.    
     
     
         3 . The method of  claim 1  or  claim 2  additionally comprising the step of: 
 (d) isolating the nucleic acid sequences encoding the ligand or receptor molecules.  
 
     
     
         4 . The method of any one of  claims 1  to  3  wherein the nucleic sequences encoding the ligand and/or receptor are associated with selection markers.  
     
     
         5 . The method of any one of the preceding claims wherein the method is used to screen a library of microorganisms displaying different ligand or receptor molecules against libraries of replicable genetic units displaying candidate specific binding partners for the ligand or receptor molecules.  
     
     
         6 . The method of any one of the preceding claims wherein the surface molecule encoding sequence of step (a) is combined with a receptor encoding sequence, or a functional fragment thereof, and the surface molecule encoding sequence of step (b) is fused to a ligand encoding sequence, or a functional fragment thereof.  
     
     
         7 . The method of any one of the preceding claims wherein the host microorganism is modified to display the ligand or receptor by a bacterial display vector.  
     
     
         9 . The method of any one of the preceding claims wherein the host microorganism is the bacteria  E. coli.    
     
     
         10 . The method of any one of the preceding claims wherein the surface molecule of the host organism is selected from the group OmpA, IgA protease or pilin.  
     
     
         11 . The method of  claim 10  wherein the surface molecule is pilin.  
     
     
         12 . The method of any one of the preceding claims wherein the replicable genetic unit is a phage display vector.  
     
     
         13 . The method of  claim 12 , wherein the phage display vector is a filamentous bacteriophage.  
     
     
         14 . The method of  claim 12  wherein the filamentous bacteriophage is fd, fl or M13.  
     
     
         15 . The method of any one of the preceding claims wherein the surface molecule of the replicable genetic unit is pIII.  
     
     
         16 . The method of any one of the preceding claims wherein the ligand is an antigen and the receptor is an antibody.  
     
     
         17 . A kit for use in carrying out a method for selection of specific ligand and receptor encoding sequences as defined in any one of  claim 1  to  16 .  
     
     
         18 . A kit for use in a method for selecting nucleic acid sequences encoding ligand and receptor molecules capable of specific binding to each other, the kit comprising: 
 (a) a host microorganism modified so that it does not display a wild type surface protein;    (b) a vector encoding said surface protein and having restriction sites for the insertion of nucleic acid encoding ligand or receptor molecules, or functional fragments thereof, so that when transformed into the host microorganism, the surface molecule and the ligand or receptor molecule are expressed as a fusion and displayed on the surface of the microorganism;    (c) a bacteriophage having a site for insertion of candidate binding partners to the ligand or receptor so that the binding partners are displayed on the surface of the bacteriophage as a fusion with a surface protein of the bacteriophage.