Recombinant Fusion Proteins and Libraries from Immune Cell Repertoires

A method of generating a T cell receptor library, comprising:

• providing a plurality of circularized polynucleotide constructs, each comprising a first polynucleotide, a second polynucleotide, and a linker polynucleotide linking the first and second polynucleotides, wherein said first polynucleotide comprises a region encoding a first variable domain from a single isolated cell, wherein said second polynucleotide comprises a region encoding a second variable domain from said single isolated cell, and wherein each of said plurality of circularized polynucleotide constructs comprises a cognate pair of linked first and second variable domains from a single isolated cell;
• inserting a third polynucleotide comprising a sequence encoding a promoter, a sequence encoding a translation initiation site, or a sequence encoding a constant region between said first and second polynucleotide in each of said circularized recombinant fusion polynucleotides, thereby generating a plurality of recombinant T cell receptor expression constructs;
• inserting said plurality of recombinant T cell receptor expression constructs into a plurality of host cells;
• and
• expressing said plurality of recombinant T cell receptor expression construct in said plurality of host cells, thereby generating a recombinant T cell receptor library comprising a plurality of recombinant T cell receptors, wherein each of said plurality of recombinant T cell receptors comprises a linked beta chain variable domain and alpha chain variable domain cognate pair from a single isolated cell.