Ligand screening and discovery

Abstract

Disclosed is a method that includes: (i) providing a plurality of initial nucleic acid cassettes that include: a) a first coding region encoding a first immunoglobulin variable domain, b) a second coding region encoding a second immunoglobulin variable domain, and c) a ribosomal binding site disposed between the first and second coding regions for translation of the second polypeptide in a first expression system, wherein the first and second coding regions are in the same translational orientation; (ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the first and second region remain physically attached during the modifying; (iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells; and (iv) expressing each modified nucleic acid cassette in the transfected cells.

1 .- 38 . (canceled) 
     
     
         39 . A method comprising:
 (i) providing a first plurality of nucleic acid cassettes that expresses a plurality of immunoglobulins in a first expression system, each nucleic acid cassette of the first plurality comprising:
 a) a first coding region encoding a first immunoglobulin variable domain; 
 b) a second coding region encoding a second immunoglobulin variable domain; and 
 c) a ribosomal binding site disposed between the first and second coding regions , 
   wherein the first and second coding regions are in the same translational orientation and are transcribed as a single transcript; and   (ii) inserting a segment between the first and second coding regions of each nucleic acid cassette of the first plurality in a single reaction mixture to produce a second plurality of nucleic acid cassettes, which expresses the plurality of immunoglobulins in a second expression system, wherein the segment comprises an internal ribosome entry site and wherein the first and second coding regions remain physically attached during the inserting step;   
       wherein one of the first and second expression systems is a prokaryotic cell and the other one is a eukaryotic cell. 
     
     
         40 . The method of  claim 39 , wherein the segment replaces the ribosomal binding site in the inserting step. 
     
     
         41 . The method of  claim 39 , wherein the prokaryotic cell is a bacterial cell. 
     
     
         42 . The method of  claim 41 , wherein the bacterial cell is  E. coli.    
     
     
         43 . The method of  claim 39 , wherein the eukaryotic cell is a yeast cell. 
     
     
         44 . The method of  claim 39 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         45 . The method of  claim 39 , wherein the first expression system is  E. coli  and the second expression system is yeast or a mammalian cell. 
     
     
         46 . The method of  claim 39 , wherein the second expression system is a mammalian cell and the segment further comprises one or more elements functional in regulating gene expression in mammalian cells, the one or more elements being selected from the group consisting of a transcriptional regulatory sequence, a chromatin control sequence, a localization signal, and a leader sequence. 
     
     
         47 . The method of  claim 46 , wherein the segment further comprises a polyA addition regulatory sequence. 
     
     
         48 . The method of  claim 39 , wherein either the first coding region or the second coding region is linked in frame to a sequence encoding an effector domain to form a fusion protein comprising the second immunoglobulin variable domain and the effector domain. 
     
     
         49 . The method of  claim 48 , wherein the effector domain comprises an immunoglobulin constant region, a polypeptide label, or a cytotoxin. 
     
     
         50 . The method of  claim 49 , wherein the effector domain comprises an immunoglobulin constant region. 
     
     
         51 . The method of  claim 50 , wherein the immunoglobulin constant region is a Fc domain. 
     
     
         52 . The method of  claim 39 , further comprising introducing the second plurality of nucleic acid cassettes into mammalian cells and express the encoded plurality of immunoglobulins in the mammalian cells. 
     
     
         53 . The method of  claim 52 , wherein the mammalian cells are cultured in a low permeability medium. 
     
     
         54 . The method of  claim 53 , wherein the mammalian cells express a heterologous protein that is attached to the cell surface and recognizes an immunoglobulin constant region, which is linked to either the first immunoglobulin variable region or the second immunoglobulin region . 
     
     
         55 . The method of  claim 54 , wherein the heterologous protein comprises an antibody specific for the immunoglobulin constant region. 
     
     
         56 . The method of  claim 54 , wherein the heterologous protein comprises an Fc receptor or an extracellular binding region thereof. 
     
     
         57 . The method of  claim 39 , further comprising introducing the second plurality of nucleic acid cassettes into yeast cells and express the encoded plurality of immunoglobulins in the yeast cells. 
     
     
         58 . The method of  claim 57 , wherein the segment further comprises a stop codon, a yeast GAL1 promoter, a yeast leader sequence, a sequence encoding an anchor protein, or a combination thereof, wherein the sequence encoding the anchor protein is in frame to either the first coding region or the second coding region. 
     
     
         59 . The method of  claim 58 , wherein the anchor protein is Aga2p. 
     
     
         60 . The method of  claim 58 , wherein the yeast leader sequence is a signal peptide for Aga2p.