Rapid iterative design of tandem-core virus-like particles using Escherichia Coli-based cell-free protein synthesis

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Rapid iterative design of tandem-core virus-like particles using Escherichia coli- based cell-free protein synthesis

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Towards the Development of a Protein Developability Selection Platform for Directed Evolution Libraries

Although the use of protein drugs has shown to be successful for the treatment of diverse diseases such as cancer and inflammatory conditions, their development still face challenges due to the unpredictability of these molecules biophysical properties in concentrated formulations for clinical use (> 100 mg/mL). Developability is defined as the capacity or suitability for development based on these biophysical properties. Low solubility, high viscosity and protein aggregation are common issues found in protein drug candidates that make the molecules suboptimal for drug development. This thesis proposes an approach for high-throughput screening of protein biophysical properties based on the principles of directed evolution that can be used in an early stage in protein drug development. Directed evolution allows to explore the effects of small random changes introduced to the candidate molecule, and selects variants enhanced in the property for which the evolution campaign was designed for. The proposed selection platform uses microfluidics to generate in vitro microcompartments, called gel-shell beads (GSBs), suitable for high- throughput selection by fluorescence-activated cell sorting (FACS) machines (~107 h-1). It also uses cell-free protein synthesis (CFPS) to fluorescently label the assessed protein by incorporation of a non-standard amino acid (nsAA) which provides a chemical group, rarely found in biological molecules, for site- specific conjugation with the fluorophore. In this thesis, a CFPS system for nsAA incorporation was generated from scratch and was adapted to be used in GSBs. Although the chosen labelling approach shown to had such non-specific binding with the GSBs that prevented a successful selection based on the label fluorescence intensity, it was still demonstrated the suitability of the CFPS-adapted GSBs to be sorted by FACS, and the DNA recovery and identification from as few as ~60 sorted GSBs. These results pave the way to higher throughput screenings for protein developability that are currently limited by use of microwell plates