Intracellular nanoparticle-based display system for high-throughput selection of protein binders

Abstract

Through the past few decades, molecular display technologies have become indispensable for creating binding proteins (binders) with desired properties. Phage display-based affinity panning and flow cytometry combined with magnetic pull-down system have been the primary methods for selecting molecular binders from combinatorial libraries. However, the marginal intensities of signal-to-noise have led to the demand for repetitive rounds of enrichment to isolate positive candidates from libraries. This process is time-consuming, error-prone, and quite laborious. Thus, there is a strong need for high signal-to-noise screening to isolate positive hits rapidly from large libraries. Inclusion bodies (IBs) are generally the result of inactive aggregation of misfolded, nonfunctional proteins, whereas some amyloid-like peptides or viral coat proteins can generate fibrous protein aggregates retaining the explicit functions of the fusion proteins. The fusion of various peptides or enzymes with a cellulose-binding domain (CBD) resulted in the formation of nanoparticles, 20？30 nm in size, with active functions. Based on this, we created a high-density protein binder library of inclusion bodies and attempted to use them as a nanoparticle-type display matrix. First, the designed variable lymphocyte receptor library (VLR), termed “Repebody,” was adopted as the binding moiety on IBs. More than 106 copies of repebody binders could be displayed on CBD particles. To interact with exogenous target molecules, the binders on IBs must be exposed to bulk solutions. For this, we attempted to crack the surface of E. coli cells, while endogenous binders and encoding DNAs are stably maintained inside cells. This technique, named “Intracellular particle display (IPD),” produced extraordinarily high signals, and resulted in the easy isolation of high-affinity binders from an immense number of negative affinity binders. Therefore, we applied the IPD system to create new affinity binders specific for different targets. The resulting binders, specific for each target, were isolated within three rounds of screening, and each round was completed in 1 day. Finally, we applied the IPD system for affinity maturation by competitive screening, resulting in the isolation of subnanomolar affinity binders (H2C13). Structural analysis of the H2C13 and sfGFP complex revealed that the binding affinity was primarily created by interactions between the C-terminal eight amino acids of sfGFP and the first half of the concave surface of the H2C13 repebody. These results proved that this method could be used to improve the binding affinity between interacting proteins, and for the isolation of new affinity binders. Herein, a novel affinity-based library screening system was developed, using nanoparticle-type IBs, to expand the utility of the IPD-based technique to other applications

two additional studies were also conducted. One was an application to isolate new metagenome-originating chaperones, which were detected by the IPD method, as it improves the interaction between target proteins on nanoparticles. As an accessary tool, the ‘Enforced Transcription’ technique, which involves the random insertion of a bidirectional T7 promoter into a metagenomic fosmid library to increase expression of metagenome-originating chaperones, was developed. The second was a novel expression system (ChroV), which expresses target genes on an F´ plasmid, to stabilize the IPD system in E. coli. In this thesis, a novel high-throughput screening system for affinity binders, and two accessary techniques, were successfully developed. When the processes were successfully combined with the IPD system, the binding moiety of the nanoparticle-type IBs could be expanded to genetically-diversified peptides, other binder proteins, or antibodies to recruit fluorescent-labeled target proteins, enzymes, or chemicals. This results in the ability to develop new beneficial functions for biotechnological applications.