Monobody is one of the promising antibody-like proteins (ALPs), which consists of approximately 100 amino-acids to form a unique β-sandwich scaffold and binds to target molecules through its loop structures with high affinity and specificity comparable to conventional IgG antibody. However, biologically produced ALPs are generally degraded by physiological proteases and therefore possibly shows immunogenicity because of their natural components of 20 proteinogenic L-amino-acids. Toward increased protease resistance and decreased immunogenicity of peptide/protein binders, several mirror-image peptides and proteins that are composed of D-amino-acids have been developed so far, based on the concept of mirror-image phage display. Since chemical protein synthesis for preparation of D-form target proteins and in vitro display technology for screening of ALPs have dramatically progressed during the past two decades, currently we will be able to create a more sophisticated system for mirror-image protein binders than the previous methods.
We have been engaged in developing new methodologies for chemical protein synthesis such as one-pot peptide ligation and thiol-free thioester synthesis. In addition, in vitro ALP screening system, called TRAP (trancription-translation coupled with association of puromycin linker) display, has been established in our group. We aimed to create mirror-image monobody against pharmaceutically important target proteins by combining TRAP display and chemical protein synthesis. D-form target proteins were prepared by chemical protein synthesis and then TRAP display was conducted to enrich monobody clones binding to the D-target with high affinity and specificity.