Beyond antibody engineering: directed evolution of alternative binding scaffolds and enzymes using yeast surface display

Abstract

Pioneered exactly 20 years ago, yeast surface display (YSD) continues to take a major role in protein engineering among the high-throughput display methodologies that have been developed to date. The classical yeast display technology relies on tethering an engineered protein to the cell wall by genetic fusion to one subunit of a dimeric yeast-mating agglutination receptor complex. This method enables an efficient genotype-phenotype linkage while exploiting the benefits of a eukaryotic expression machinery. Over the past two decades, a plethora of protein engineering efforts encompassing conventional antibody Fab and scFv fragments have been reported. In this review, we will focus on the versatility of YSD beyond conventional antibody engineering and, instead, place the focus on alternative scaffold proteins and enzymes which have successfully been tailored for purpose with regard to improving binding, activity or specificity.

Engineering repeat proteins of the immune system

“…Based on repeat protein domains, these proteins are simple to design and modular, with highly variable binding surfaces. Compared with immunoglobulins, these proteins offer high affinity and specificity for their targets . While many protein engineering efforts are only able to target proteins and peptides, these engineered immune proteins are able to target glycans as well, filling an important gap in tools for research and biomedical applications.…”

“…Immunoglobulin (Ig) antibodies are the best‐studied examples of such recognition agents, and have been reviewed extensively . However, problems with stability, expense of expression, and specificity of interaction of Ig antibodies have led in recent years to the development of alternative binding scaffolds . Antibodies rely on disulfide bond formation for their stability, requiring difficult, expensive eukaryotic cell production; this also makes them unsuitable for applications in reducing conditions .…”

“…In the search for better recognition agents, many different scaffolds have emerged; some notable examples are peptide and DNA aptamers, affimers, and anticalins, among others. The motivation and results of this research have been extensively reviewed in recent years . among these scaffolds, repeat proteins such as designed ankyrin repeat proteins (DARPins) tetratricopeptide repeat proteins (TPRs), and armadillo repeats have emerged as a promising class of scaffolds.…”

Directed Evolution of a Bond‐Forming Enzyme: Ultrahigh‐Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display

“…To conclude, in the present work we added microbial transglutaminase from Streptomyces mobaraensis to the list of enzymes whose catalytic activity can be improved upon engineering using yeast surface display . However, the screening of enzymes, especially the bond‐forming ones, by YSD remains challenging because an acceptor molecule within the range of the surface‐displayed enzyme is required.…”

Ultrahigh‐throughput screening system for directed polymer binding peptide evolution

“…Display of peptides on Escherichia coli ( E. coli ) or yeast cell surfaces are alternative systems often used in directed evolution campaigns (van Bloois, Kolmar, & Fraaije, ) due to their general applicability and simplicity in handling. Both, antibodies as wells as scaffold proteins have been engineered applying yeast display technologies as summarized in two comprehensive reviews by Boder, Raeeszadeh‐Sarmazdeh and Price () and Konning and Kolmar (). Yeast display systems commonly contain library sizes of up to 10 9 mutants, but are comparably time‐consuming due to the growth rate of yeast cells (Benatuil, Belk, & Hsieh, ; Salema & Fernandez, ).…”