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Yeast surface display is a versatile protein engineering technology, enabling precise control of the applied selection pressure. We present a yeast-surface-display-based protocol for the enrichment of binders specifically recognizing ligand-bound receptors. We describe steps for magnetic bead selections, random mutagenesis, and flow cytometric sorting, followed by library sequencing and detailed analysis of enriched clones. While this approach is exemplified with rcSso7d-based libraries and epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) complexes, it can also be adapted to other binder scaffolds and ligand-receptor systems. For complete details on the use and execution of this protocol, please refer to Dobersberger et al. 1 ., Competing Interests: Declaration of interests M.W.T. receives funding from Miltenyi Biotec., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

In addition to its biological function, the stability of a protein is a major determinant for its applicability. Unfortunately, engineering proteins for improved functionality usually results in destabilization of the protein. This so-called stability-function trade-off can be explained by the simple fact that the generation of a novel protein function─or the improvement of an existing one─necessitates the insertion of mutations, i.e. , deviations from the evolutionarily optimized wild-type sequence. In fact, it was demonstrated that gain-of-function mutations are not more destabilizing than other random mutations. The stability-function trade-off is a universal phenomenon during protein evolution that has been observed with completely different types of proteins, including enzymes, antibodies, and engineered binding scaffolds. In this review, we discuss three types of strategies that have been successfully deployed to overcome this omnipresent obstacle in protein engineering approaches: (i) using highly stable parental proteins, (ii) minimizing the extent of destabilization during functional engineering (by library optimization and/or coselection for stability and function), and (iii) repairing damaged mutants through stability engineering. The implementation of these strategies in protein engineering campaigns will facilitate the efficient generation of protein variants that are not only functional but also stable and therefore better-suited for subsequent applications.

T cells that are genetically engineered to express chimeric antigen receptors (CAR T cells) have shown impressive clinical efficacy against B-cell malignancies. In contrast to these highly potent CD19-targeting CAR T cells, many of those directed against other tumor entities and antigens currently suffer from several limitations. For example, it has been demonstrated that many scFvs used as antigen-binding domains in CARs show some degree of oligomerization, which leads to tonic signaling, T cell exhaustion, and poor performance in vivo. Therefore, in many cases alternatives to scFvs would be beneficial. Fortunately, due to the development of powerful protein engineering technologies, also non-immunoglobulin-based scaffolds can be engineered to specifically recognize antigens, thus eliminating the historical dependence on antibody-based binding domains. Here, we discuss the advantages and disadvantages of such engineered binding scaffolds, in particular with respect to their application in CARs. We review recent studies, collectively showing that there is no functional or biochemical aspect that necessitates the use of scFvs in CARs. Instead, antigen recognition can also be mediated efficiently by engineered binding scaffolds, as well as natural ligands or receptors fused to the CAR backbone. Finally, we critically discuss the risk of immunogenicity and show that the extent of nonhuman amino acid stretches in engineered scaffolds-even in those based on nonhuman proteins-is more similar to humanized scFvs than might be anticipated. Together, we expect that engineered binding scaffolds and natural ligands and receptors will be increasingly used for the design of CAR T cells., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)