John Hancock, Brigham Young University
Life Sciences
LLO56 and LLO118 are CD4+ T cells specific for the same Listeria monocytogenes epitope. Despite their TCRs differing by only 15 amino acids, LLO118 and LLO56 have dramatically different primary and secondary responses to Listeria monocytogenes infection. We reasoned that LLO56, the single chain TCR (Vβ2-linker-Vα2) could be subjected to directed evolution to generate mutants that are more stable and bind to peptide-MHC with higher affinity. Single chain LLO56 was fused to the yeast surface protein Aga-2 and error prone PCR was used to generate mutagenic libraries. A first generation stabilized single chain TCR (scTCR) was selected using biotinylated Vβ2 and Vα2 antibodies and anti-biotin beads. The first generation LLO56 mutant expressed LLO56 on the surface of yeast at higher levels than wild type by flow cytometry. To produce mutants with additional stability, a second-generation mutant was generated by combining multiple stability mutations isolated in a number of first generation clones.