Surface expression libraries of heteromeric receptors

Abstract

A composition of matter comprising a plurality of procaryotic cells containing diverse combinations of first and second DNA sequences encoding first and second polypeptides which form a heteromeric receptor exhibiting binding activity toward a preselected molecule, said heteromeric receptors being expressed on the surface of filamentous bacteriophage.

1 . A composition of matter comprising a plurality of cells containing diverse combinations of first and second DNA sequences encoding first and second polypeptides which form heteromeric receptors, one or both of said polypeptides being expressed as fusion proteins on the surface of a cell.  
     
     
         2 . The composition of  claim 1 , wherein said plurality of cells are  E. coli.    
     
     
         3 . The composition of  claim 1 , wherein said heteromeric receptors selected from the group consisting of antibodies, T cell receptors, integrins, hormone receptors and transmitter receptors.  
     
     
         4 . The composition of  claim 1 , wherein said first and second DNA sequences encode functional portions of heteromeric receptors.  
     
     
         5 . The composition of  claim 4 , wherein said first and second DNA sequences encode functional portions of the variable heavy and variable light chains of an antibody.  
     
     
         6 . The composition of  claim 1 , wherein said cell produces filamentous bacteriophage.  
     
     
         7 . The composition of  claim 6 , wherein said filamentous bacteriophage are selected from the group consisting of M13, fd and fl.  
     
     
         8 . The composition of  claim 6 , wherein at least one of the encoded first or second polypeptides is expressed as a fusion protein with gene VIII.  
     
     
         9 . A kit for the preparation of vectors useful for the coexpression of two or more DNA sequences encoding polypeptides which form heteromeric receptors comprising two vectors, a first vector having two pairs of restriction sites symmetrically oriented about a cloning site which can be combined with a second vector, having two pairs of restriction sites symmetrically oriented about a cloning site and in an identical orientation to that of the first vector, wherein one or both vectors contains sequences necessary for expression of polypeptides encoded by DNA sequences inserted in said cloning sites.  
     
     
         10 . The kit of  claim 9 , wherein said first and second vectors are circular.  
     
     
         11 . The kit of  claim 9 , wherein said expression peptides is as fusion proteins on the surface of a cell.  
     
     
         12 . The kit of  claim 9 , wherein said cell produces filamentous bacteriophage.  
     
     
         13 . The kit of  claim 9 , wherein said filamentous bacteriophage is selected from the group consisting of M13, fd and fl.  
     
     
         14 . The kit of  claim 13 , wherein at least one of the DNA sequences is expressed as a fusion protein with gene VIII.  
     
     
         15 . The kit of  claim 9 , wherein said two pairs of restriction sites are Hind III-Mlu I and Hind III-Mlu I.  
     
     
         16 . A cloning system for the coexpression of two or more DNA sequences encoding polypeptides which form a heteromeric receptor, comprising a set of first vectors having a diverse population of first DNA sequences and a set of second vectors having a diverse population second DNA sequences, said first and second vectors having two pairs of restriction sites symmetrically oriented about a cloning site for containing said first and second populations of DNA sequences so as to allow only the operational combination of vector sequences containing said first and second DNA sequences.  
     
     
         17 . The cloning system of  claim 16 , wherein said first and second vectors are circular.  
     
     
         18 . The cloning system of  claim 16 , wherein said heteromeric receptors selected from the group consisting of antibodies, T cell receptors, integrins, hormone receptors and transmitter receptors.  
     
     
         19 . The cloning system of  claim 16 , wherein said first and second DNA sequences encode functional portions of heteromeric receptors.  
     
     
         20 . The cloning system of  claim 19 , wherein said first and second DNA sequences encode functional portions of the variable heavy and variable light chains of an antibody.  
     
     
         21 . The cloning system of  claim 16 , wherein said coexpression of two or more DNA sequences encoding polypeptides which form a heteromeric receptor is on the surface of cell.  
     
     
         22 . The cloning system of  claim 16 , wherein said cell produces a filamentous bacteriophage.  
     
     
         23 . The cloning system of  claim 22  wherein said filamentous bacteriophage selected from the group consisting of M13, fd and fl.  
     
     
         24 . The cloning system of  claim 23 , wherein at least one of the DNA sequences is expressed as a fusion protein with the protein product of gene VIII.  
     
     
         25 . The cloning system of  claim 16 , wherein said two pairs of restriction sites are Hind III-Mlu I and Hind III-Mlu I.  
     
     
         26 . A plurality of expression vectors containing a plurality of possible first and second DNA sequences encoding polypeptides which form a heteromeric receptor exhibiting binding activity toward a preselected molecule, said DNA sequence encoding heteromeric receptors being operatively linked to genes encoding surface proteins of a cell.  
     
     
         27 . The expression vectors of  claim 26 , wherein said expression vectors are circular.  
     
     
         28 . The expression vectors of  claim 23 , wherein said heteromeric receptors are selected from the group consisting of antibodies, T cell receptors, integrins, hormone receptors and transmitter receptors.  
     
     
         29 . The expression vectors of  claim 26 , wherein said first and second DNA sequences encode functional portions of heteroeric receptors.  
     
     
         30 . The expression vectors of  claim 29 , wherein said first and second DNA sequences encode functional portions of the variable heavy and variable light chains of an antibody.  
     
     
         31 . The expression vectors of  claim 26 , wherein said cells produce filamentous bacteriophage.  
     
     
         32 . The expression vectors of  claim 26 , wherein said filamentous bacteriophage are selected from the group consisting of M13, fd and fl.  
     
     
         33 . The expression vectors of  claim 32 , wherein at least one of the encoded first or second polypeptides is expressed as a fusion protein with gene VIII.  
     
     
         34 . A method of constructing a diverse population of vectors capable of expressing a diverse population of heteromeric receptors, comprising: 
 (a) operationally linking to a first vector a first population of diverse DNA sequences encoding a diverse population of first polypeptides, said first vector having two pairs of restriction sites symmetrically oriented about a cloning site;    (b) operationally linking to a second vector a second population of diverse DNA sequences encoding a diverse population of second polypeptides, said second vector having two pairs of restriction sites symmetrically oriented about a cloning site in an identical orientation to that of the first vector; and    (c) combining the vector products of step (a) and (b) under conditions which allow only the operational combination of vector sequences containing said first and second DNA sequences.    
     
     
         35 . The method of  claim 34 , wherein said first and second vectors are circular.  
     
     
         36 . The method of  claim 34 , wherein said heteromeric receptors are selected from the group consisting of antibodies, T cell receptors, integrins, hormone receptors and transmitter receptors.  
     
     
         37 . The method of  claim 34 , wherein said first and second DNA sequences encode functional portions of the variable heavy and variable light chains of an antibody.  
     
     
         38 . The method of  claim 34 , wherein said expression of a diverse population of heteromeric receptors is on the surface of a cell.  
     
     
         39 . The method of  claim 37 , wherein said cell produces a bacteriophage.  
     
     
         40 . The method of  claim 39 , wherein said filamentous bacteriophage is selected from the group consisting of M13, fd and fl.  
     
     
         41 . The method of  claim 34 , wherein at least one of said first or second DNA sequences is expressed as a gene VIII fusion protein.  
     
     
         42 . The method of  claim 34 , wherein said two pairs of restriction sites are Hind III-Mlu I and Hind III-Mlu I.  
     
     
         43 . The method of  claim 34 , wherein said combining step further comprises: 
 (C1) restricting said first vector with a restriction enzyme recognizing one of the restriction sites encoded in said two pairs of restriction sites;    (C2) restricting said second vector with a different restriction enzyme recognizing the second restriction site encoded in said two pairs of restriction sites;    (C3) digesting the 3′ ends of said restricted first and second vectors with an exonuclease; and    (C4) annealing said first and second vectors.    
     
     
         44 . A method for selecting a heteromeric receptor exhibiting binding activity toward a preselected molecule from a population of diverse heteromeric receptors, comprising: 
 (a) operationally linking to a first vector a first population of diverse DNA sequences encoding a diverse population of first polypeptides, said first vector having two pairs of restriction sites symmetrically oriented about a cloning site;    (b) operationally linking to a second vector a second population of diverse DNA sequences encoding a diverse population of second polypeptides, said second vector having two pairs of restriction sites symmetrically oriented about a cloning site in an identical orientation to that of the first vector;    (c) combining the vector products of step (a) and (b) under conditions which allow only the operational combination of vector sequences containing said first and second DNA sequences.    (d) introducing said population of combined vectors into a compatible host under conditions sufficient for expressing said population of first and second DNA sequences; and    (e) determining the heteromeric receptors which bind to said preselected molecule.    
     
     
         45 . The method of  claim 44 , wherein said first and second vectors are circular.  
     
     
         46 . The method of  claim 44 , wherein said heteromeric receptors are selected from the group consisting of antibodies, T cell receptors, integrins, hormone receptors and transmitter receptors.  
     
     
         47 . The method of  claim 44 , wherein said first and second DNA sequences encode functional portions of heteromeric receptors.  
     
     
         48 . The method of  claim 47 , wherein said first and second DNA sequences encode functional portions of the variable heavy and variable light chains of an antibody.  
     
     
         49 . The method of  claim 44 , wherein said expression of a diverse population of heteromeric receptors is on the surface of a cell.  
     
     
         50 . The method of  claim 49 , wherein said cell produces a filamentous bacteriophage.  
     
     
         51 . The method of  claim 50 , wherein said filamentous bacteriophage is selected from the group consisting of M13, fd and fl.  
     
     
         52 . The method of  claim 51 , wherein at least one of said first or second DNA sequences is expressed as a gene VIII fusion protein.  
     
     
         53 . The method of  claim 44 , wherein said two pairs of restriction sites are Hind III-Mlu I and Hind III-Mlu I.  
     
     
         54 . The method of  claim 44 , wherein said combining step further comprises: 
 (C1) restricting said first vector with a restriction enzyme recognizing one of the restriction sites encoded in said two pairs of restriction sites;    (C2) restricting said second vector with a different restriction enzyme recognizing the second restriction site encoded in said two pairs of restriction sites;    (C3) digesting the 3′ ends of said restricted first and second vectors with an exonuclease; and    (C4) annealing said first and second vectors.    
     
     
         55 . A method for determining the nucleic acid sequences encoding a heteromeric receptor exhibiting binding activity toward a preselected molecule from a diverse population of heteromeric receptors, comprising: 
 (a) operationally linking to a first vector a first population of diverse DNA sequences encoding a diverse population of first polypeptides, said first vector having two pairs of restriction sites symmetrically oriented about a cloning site;    (b) operationally linking to a second vector a second population of diverse DNA sequences encoding a diverse population of second polypeptides, said second vector having two pairs of restriction sites symmetrically oriented about a cloning site in an identical orientation to that of the first vector;    (c) combining the vector products of step (a) and (b) under conditions which allow only the operational combination of vector sequences containing said first and second DNA sequences.    (d) introducing said population of combined vectors into a compatible host under conditions sufficient for expressing said population of first and second DNA sequences;    (e) determining the heteromeric receptors which bind to said preselected molecule;    (f) isolating the nucleic acid sequences 5 encoding said first and second polypeptides; and    (g) sequencing said nucleic acid sequences.    
     
     
         56 . The method of  claim 55 , wherein said first and second vectors are circular.  
     
     
         57 . The method of  claim 55 , wherein said first heteromeric receptors selected from the group consisting of antibodies, T cell receptors, integrins, hormone receptors and transmitter receptors.  
     
     
         58 . The method of  claim 55 , wherein said first and second DNA sequences encode functional portions of heteromeric receptors.  
     
     
         59 . The method of  claim 58 , wherein said first and second DNA sequences encode functional portions of the variable heavy and variable light chains of an antibody.  
     
     
         60 . The method of  claim 55 , wherein said expression of a diverse population of heteromeric receptors is on the surface of a cell filamentous bacteriophage selected from the group consisting of M13, fd and fl and at least one of said first or second DNA sequences is expressed as a gene VIII fusion protein.  
     
     
         61 . The method of  claim 55 , wherein said cell produces filamentous bacteriophage.  
     
     
         62 . The method of  claim 61 , wherein said filamentous bacteriophage is selected from the group consisting of M13, fd and fl.  
     
     
         63 . The method of  claim 62 , wherein at least one of said frist or second DNA sequences is expressed as a gene VIII fusion protein.  
     
     
         64 . The method of  claim 50 , wherein said two pairs of restriction sites are Hind III-Mlu I and Hind III-Mlu I.  
     
     
         65 . The method of  claim 50 , wherein said combining step further comprises: 
 (C1) restricting said first vector with a restriction enzyme recognizing one of the restriction sites encoded in said two pairs of restriction sites;    (C2) restricting said second vector with a different restriction enzyme recognizing the second restriction site encoded in said two pairs of restriction sites;    (C3) digesting the 3′ ends of said restricted first and second vectors with an exonuclease; and    (C4) annealing said first and second vectors.    
     
     
         66 . A vector comprising two copies of a gene encoding a filamentous bacteriophage coat protein, one copy of said gene capable of being operationally linked to a DNA sequence encoding a polypeptide of a heteromeric receptor wherein said DNA sequence can be expressed as a fusion protein on the surface of said filamentous bacteriophage or as a soluble polypeptide.  
     
     
         67 . The vector of  claim 66 , wherein said two copies of said gene encode substantially the same amino acid sequence but have different nucleotide sequences.  
     
     
         68 . The vector of  claim 66 , wherein said one copy of said gene is expressed on the surface of said filamentous bacteriophage.  
     
     
         69 . The vector of  claim 66 , wherein said bacteriophage coat protein is M13 gene VIII.  
     
     
         70 . The vector of  claim 66 , wherein said vector has substantially the same sequence as that shown in  FIG. 2  (SEQ ID NO: 1)  
     
     
         71 . A vector comprising sequences necessary for the coexpression of two or more inserted DNA sequences encoding polypeptides which form heteromeric receptors and two copies of a gene encoding a filamentous bacteriophage coat protein, one copy of said gene capable of being operationally linked to one of said two or more inserted DNA sequences wherein said DNA sequence can be expressed as a fusion protein on the surface of said filamentous bacteriophage or as a soluble polypeptide.  
     
     
         72 . The vector of  claim 71 , wherein said two copies of said gene encode substantially the same amino acid sequence but have different nucleotide sequences.  
     
     
         73 . The vector of  claim 71 , wherein said one copy of said gene is expressed on the surface of said filamentous bacteriophage.  
     
     
         74 . The vector of  claim 71 , wherein said bacteriophage coat protein is M13 gene VIII.  
     
     
         75 . The vector of  claim 71 , wherein said vector has substantially the same sequence as that shown in  FIG. 6  (SEQ ID NO: 5).