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Exploring multivalent technologies and strategies for directed protein evolution



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Koch, Wolfgang 


Evolution is the continuous adaptation to ever changing environments and pressure for survival of all living species. Directed evolution attempts to mimic this process of natural selection of biological molecules and systems towards a specific function that would otherwise remain untapped by nature. For this purpose, it uses iterative rounds of mutation and artificial selection to improve existing or generate entirely new biomolecules. However, a series of stepwise improvements during the selection process can lead to local entrapments in the respective sequences and fitness, and thus abolish access to further phenotypic diversity. Multivalency is a strategy commonly applied by nature that has the potential to increase the strength of interaction and consequently the recovery and fitness of variants. As a result, this strategy may be able to support and tolerate mutations that are negative in one context but can become beneficial in others during the course of evolution. This thesis aims to characterize and investigate the effects of multivalency on the outcomes and intermediate states of evolution campaigns. To test the hypothesis of increased survival of variants by introduction of multivalency during the selection process, a new in vitro multivalent selection system was developed, that allowed for the display of proteins of interest under defined valencies. Using this system, different valency effects could be quantified and mapped. The observations of vastly improved recoveries and enrichments of variants by multivalent display suggests that the introduction of multivalency in a selection system can substantially alter the stringency of selection pressure on variants, while maintaining positive enrichment of functional variants during the course of a selection campaign. Based on this confirmed hypothesis, two main questions were addressed: Do identical naïve libraries, subjected to different evolutionary stringencies by multivalency, evolve a common set of amino acids and do these naïve libraries, submitted to different evolutionary stringencies, evolve different fitness optima? To address these questions, a new phage display system was developed that enabled the display of variants under a range of defined valencies and allowed for the creation of large (>109) naïve phage-libraries of Designed Ankyrin Repeat Proteins (DARPins). The resulting libraries were subjected to evolution campaigns and the evolved variants emerging from selection rounds, as well as intermediate states, analyzed to address the questions of whether differences in trajectories in sequence space towards fitness optima can be identified. A third section of this thesis focused on the effects of high selection pressure on the evolution of weak promiscuous activities. For these investigations, I evolved and characterized proteins with binding specificities towards a range of pathogenic human coronaviruses. As such, insight into the potential of occupied sequence space with overlapping fitness optima towards different distantly related targets for the subsequent evolution of broadly specific interactions could be obtained. The tools established and insights obtained in this thesis can provide an improved understanding of the effects of multivalency and selection stringency on the outcomes and intermediate states of evolution campaigns. As such, these novel insights contribute to a more comprehensive understanding of the underlying principles, that may govern the outcome of directed evolution campaigns of proteins.





Hollfelder, Florian
Jermutus, Lutz


directed evolution, multivalency, phage display, snap display, fitness landscape


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
European Commission Horizon 2020 (H2020), Marie Skłodowska-Curie Actions (722610)