Recombinant Fusion Proteins and Libraries from Immune Cell Repertoires
Disclosed herein are methods and compositions for generating a repertoire of recombinant fusion polypeptides from immune cells, and uses thereof.
1 . A method for preparing a plurality of recombinant immunoglobulin expression constructs, comprising:
providing a plurality of recombinant fusion polynucleotides each comprising a first polynucleotide encoding a first variable domain, a second polynucleotide encoding a second variable domain, and a linker polynucleotide linking the first and second polynucleotides;
expressing a plurality of recombinant fusion polypeptides encoded by said plurality of recombinant fusion polynucleotides to display said plurality of recombinant fusion polypeptides on a plurality of surfaces;
enriching said plurality of recombinant fusion polynucleotides for binding to an antigen by exposing said plurality of surfaces to said antigen and selecting based on binding between said antigen and each of said plurality of recombinant fusion polypeptides;
circularizing one or more of said enriched recombinant fusion polynucleotides; and
inserting a third polynucleotide comprising a sequence encoding a promoter and a sequence encoding a constant region between said first and second polynucleotide in each of said circularized enriched recombinant fusion polynucleotides, thereby generating a plurality of recombinant immunoglobulin expression constructs.
2 . The method of claim 1 , further comprising:
inserting said plurality of recombinant immunoglobulin expression constructs into a plurality of host cells; and
expressing said plurality of recombinant immunoglobulin expression construct in said plurality of host cells, thereby generating a recombinant immunoglobulin library, wherein each recombinant immunoglobulin comprises a linked heavy chain and light chain variable domain cognate pair from a single isolated cell.
3 . The method of claim 1 , wherein said first and second variable domains are from a single cell.
4 . The method of claim 1 , wherein said first variable domain is from an immunoglobulin heavy chain, and said second variable domain is from an immunoglobulin light chain.
5 . The method of claim 1 , wherein said plurality of recombinant immunoglobulin expression constructs comprises at least 1,000, 10,000, or 100,000 unique cognate pairs of heavy chain and light chain encoding sequences.