Periodic Reporting for period 1 - FUNC NGS (Deep screening of proteins with a next generation sequencing platform)
Periodo di rendicontazione: 2015-08-01 al 2017-07-31
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.
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.