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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - FUNC NGS (Deep screening of proteins with a next generation sequencing platform)

Teaser

Summary

Protein engineering is often required to improve the efficiency of protein-based therapeutic agents. Despite the remarkable advancements in computer-aided protein design, the most potent candidates for a binding interaction are still found by experimental methods. The analysis of more than a few hundred to thousand individual variants requires automated high-throughput screening platforms, but the throughput of even the most modern microtiter plate screening methods is still a tiny fraction of the size of combinatorial repertoires operated with phage and ribosome display (>109). Therefore, better assays for quantitative protein interaction analysis are still highly demanded. Next generation sequencing (NGS) has become an indispensable tool in visualizing the effect of mutations in proteins on a global scale using hundreds of millions of dna sequences. Several chemistries have been developed for NGS, of which, the most used today are Ion Torrent semiconductor sequencing technology, Roche 454 pyrosequencing and Illumina sequencing by synthesis technology.
The objective of this proposal was to set up a generalized method for analysing protein interactions using a cell-free display technique combined to a next-generation sequencing platform in order to increase the throughput of protein interaction analysis. A method was designed that used microbeads and protein DNA conjugates as the media for protein reactions. The technology was aimed to be compatible with most next generation sequencing platforms for analysis. Research towards the final deep screening technology was supported by other technology development projects targeting different aspects of bead surface display methods.

Work performed

During the fellowship a robust microbead surface display technique was developed in collaboration with other co-workers that was compatible with the deep screening platform. The developed cell-free bead platform was based on a novel on-bead DNA assembly technique, which has not been explored in prior art. Protocols for preparing the bead repertoires were optimized and novel bead coupling chemistries were established for orthogonal tethering of DNA and protein on the beads. The developed technology was characterized in bead library selections using fluorescently labeled target molecule and flow cytometric sorting to enrich target binding molecules from the population.
The feasibility of the deep screening platform, named PIDA (protein interaction dependent assembly), was studied using SpyCatcher/SpyTag binder pair as the model system. Spycatcher and SpyTag are parts of a split bacterial adhesin. The two parts form a covalent bond with each other upon association. Major part of the work aiming towards PIDA-assay was to design DNA recovery methods for the unambiguous amplification of correctly assembled DNA avoiding unspecific background amplification. After optimization of the reaction conditions, correct pairing of SpyCatcher-DNA and SpyTag-DNA on the surface of microbeads was verified by agarose gel analysis and sequencing confirming the feasibility of the PIDA-technology. SpyCatcher-DNA paired correctly with the target peptide, i.e., SpyTag, with 75% efficiency when non-target peptides were provided in the same reaction. It was also observed that polyethylene glycol could be used to improve the interaction specificity. Due to technical challenges intrinsic to the method and the limited time frame the work was focused on optimizing the proof-of-concept experiments. Consequently, the benefits of PIDA-assay in comparison to other techniques in the art remain to be shown in large scale library studies.
In parallel to the sequencing-based PIDA-assay, an analogous microbead platform was developed, based on optical signal analysis with flow cytometry. The developed color-coded microbead screening platform was applied to screening anti-digoxigenin ScFv clone repertoires for affinity-improved clones. Ten-fold improvement in digoxigenin binding affinity was obtained by the optical bead screening platform. Manuscripts on the novel bead surface display method using on-bead DNA assembly and the color-coded suspension bead array technology have been prepared to be submitted for peer-review.

Final results

In this project I explored a novel technology operating at the interface of protein engineering and next generation sequencing. The developed technique, PIDA-assay, would require further optimization to be sufficiently robust to challenge the performance of the established screening techniques in industrial bioaffinity reagent development. During my project other laboratories published similar protein interaction technologies demonstrating the shared interests in harnessing the potential of the next generation sequencing technique in protein engineering. The application areas of the deep screening techniques are in protein-based therapy research, diagnostics and synthetic biology. At the matured stage simple robust technologies at this interface enable all scientists to answer complex biological questions with minimal budgets. The work towards the final goal lead also to the discovery and characterization of two microbead based platforms that demonstrate technological advancements to the currently available methods and will hopefully be widely applied by other scientists to advance protein engineering science.