Xenoprotein engineering via synthetic libraries

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means.

Enumeration of human antigen–specific naive CD8+ T cells reveals conserved precursor frequencies

The number of antigen-specific naive CD8+ T cells is believed to be important in the shaping of adaptive immune responses, and is predictive for the magnitude of priming responses in mouse models. Because of extremely low precursor frequencies, knowledge about these cells comes from indirect techniques and estimations. Here, we present a strategy based on the combination of tetramer staining, magnetic-bead enrichment, and multiparametric cytometry, which permitted direct detection and analysis of CD8+ T cells reactive for 6 different naive epitopes (MART-126-35, HIV-1 Gag p1777-85, hepatitis C virus [HCV] NS31406-1415, HCV Core132-140, NY-ESO-1157-165, and cytomegalovirus [CMV] pp65495-503). Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6 × 10−6 to 1.3 × 10−4), but conserved frequencies among humans. Development of a procedure for direct assessment of T-cell precursor frequency in humans has important implications, with particular relevance to vaccine development and monitoring of tumor and self-reactive T cells.