Construction and functional validation of combinatorial designed ankyrin repeat protein (DARPin) libraries for phage display

Abstract

Designed ankyrin repeat protein (DARPin) is one of the most advanced alternative scaffold proteins. In this study, a combinatorial DARPin library for phage display was constructed. Firstly, a binary DARPin phage display library (termed BiLiBST) was constructed containing variating tyrosine and serine residues. Five PelB signal sequence mutants were then analyzed to find the one with the best display levels for BiLiBST. These variants were found in earlier study on anti-GFP DARPin display using modified signal sequence, but it was not known if the increased display levels were a generic behavior irrespective of displaying either fixed DARPin protein or library DARPin. The capability of each PelB variant to display BiLiBST was determined by anti-DARPin immunoassay, and the phage stocks titers were normalized by total phage immunoassay. All tested PelB variants exhibited statistically significant improvement in display of the BiLiBST compared to the parental PelB and few variants had even twofold higher display efficiency than DsbA, which has previously been the standard for filamentous phage display of DARPins. The BiLiBST library was displayed with DN5 signal sequence and preselected for open reading frames by panning two rounds against anti-DARPin Fabs. The DARPin libraries were effectively purified from frameshifts as the frequency of frameshift decreased from 50 % to 13 %. The main DARPin library construction was performed by assembly PCR using purified BiLiBST as template and its validity was confirmed by selecting against the GST-tagged protein N from SARS-COV-2 for three rounds. Successful enrichment of binders was confirmed by phage immunoreactivity assay with signal to background ratios up to 11-fold. This is a solid foundation for further molecular diversification and library construction.