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Professor Carl Denard, University of Florida
Tuesday, September 28, 2021, 11:00am - 12:00pm

Carl DenardDeciphering and Engineering the Substrate Specificity of Protein-Modifying Enzymes

Protein-modifying enzymes (PMEs) are ubiquitous in biology, playing significant roles in initiating, regulating, and terminating cellular processes. The diversity and breadth of their substrate preference have been harnessed for a variety of applications, including protease therapeutics, protein purification, mass spectrometry-based proteomics, targeted therapeutics, and site-specific bioconjugations. Furthermore, with advances in protein engineering and synthetic biology, they are now being utilized for studying protein-protein interactions, imaging newly synthesized proteins, and performing logic operations inside cells. To successfully repurpose PMEs towards these applications, it is often necessary to engineer their catalytic properties and to profile their substrate specificities. In searching for desired activities in a large pool of variants, a high-throughput screening or selection system should ideally exhibit a broad operational range (dose-response or sensitivity over a large variation in input), and a high dynamic range (signal-to-noise ratio).

Here we describe YESS 2.0, a highly modular and customizable yeast endoplasmic sequestration screening system suitable for engineering and profiling the specificity of protein-modifying enzymes. By incorporating features to modulate gene transcription, as well as substrate and enzyme spatial sequestration within a versatile and seamless assembly method, YESS 2.0 achieves broad operational and dynamic range. To showcase YESS 2.0, we evolve a TEV protease variant (eTEV) with an 8-fold higher catalytic efficiency to obtain the fastest TEV protease variant to date. Due to the unique features of our system, this phenotype is strictly attributable to an increase in turnover number (kcat). Second, we use YESS 2.0 coupled with NextGen Sequencing to profile the substrate specificity of insulin-degrading enzyme (IDE) and discover IDE substrates with highly increased activity compared to the prototypical insulin peptide. Lastly, we show for the first time that YESS 2.0 supports calcium-independent sortase-mediated ligations (SML) and confirm that residues directly C-terminal of the pentapeptide motif heavily influence the rate of SML. This state-of-the-art platform offers unmatched versatility in profiling and engineering the specificity of protein-modifying enzymes and should enable even more ambitious future undertakings.

Hosted by Professor Sagar Khare

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