Development of a Split Esterase for Protein-Protein Interaction Dependent Small Molecule Activation

Kentala, Kaitlin; Beck, Michael; An, Wei; Lippert, Alexander R.; Lewis, Jared C.; Dickinson, Bryan C.

doi:10.26434/chemrxiv.8258633.v2

Cited by 9 publications

(9 citation statements)

References 33 publications

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“…This modular strategy may be applied to diverse functional proteins to control bioluminescence 5 , 6 , fluorescence 7 , proteolytic cleavage 8 - 10 and transcription 11 , 12 . As a result, conditionally reconstituted split proteins have been employed in a variety of applications including probing and discovering new protein-protein interactions 13 - 16 , studying post-translational modifications 17 , establishing small molecule-regulated control over enzymatic activity 18 , 19 , and rewiring cellular signaling 9 , 10 .…”

Section: Introductionmentioning

confidence: 99%

Computation-guided optimization of split protein systems

et al. 2021

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Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. This new technology will streamline the generation and use of split protein systems for diverse applications.

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Section: Introductionmentioning

confidence: 99%

Computation-guided optimization of split protein systems

et al. 2021

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“…To establish the feasibility of improving BS2 via PACS using the ABA biosensor, a library of BS2 variants was generated from WT BS2 and the four PACE variants noted above via error prone PCR with a high error rate to ensure sampling of as many unique variants as possible 61 (Figure S10). Aliquots of library phage were cultured with an ester-caged fluorescein 62,63 to confirm that the library contained active variants, and significant fluorescence was observed (Figure S11). Biocatalyst PACS, which is identical to the PACE system except omitting the mutagenesis plasmid, was then initiated with the BS2 library and 4b to enrich for variants with improved activity on 4b.…”

Section: ■ Resultsmentioning

confidence: 99%

Phage-Assisted Continuous Evolution and Selection of Enzymes for Chemical Synthesis

et al. 2021

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Ligand-dependent biosensors are valuable tools for coupling the intracellular concentrations of small molecules to easily detectable readouts such as absorbance, fluorescence, or cell growth. While ligand-dependent biosensors are widely used for monitoring the production of small molecules in engineered cells and for controlling or optimizing biosynthetic pathways, their application to directed evolution for biocatalysts remains underexplored. As a consequence, emerging continuous evolution technologies are rarely applied to biocatalyst evolution. Here, we develop a panel of ligand-dependent biosensors that can detect a range of small molecules. We demonstrate that these biosensors can link enzymatic activity to the production of an essential phage protein to enable biocatalyst-dependent phage-assisted continuous evolution (PACE) and phage-assisted continuous selection (PACS). By combining these phage-based evolution and library selection technologies, we demonstrate that we can evolve enzyme variants with improved and expanded catalytic properties. Finally, we show that the genetic diversity resulting from a highly mutated PACS library is enriched for active enzyme variants with altered substrate scope. These results lay the foundation for using phage-based continuous evolution and selection technologies to engineer biocatalysts with novel substrate scope and reactivity.

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“…78−80 We also developed chemiluminescent reporter Chemilum-CM to complement a BS2 split-esterase system for monitoring cellular protein−protein interactions (Figure 12A). 81 The ester-masked probe generated chemiluminescence response upon rapamycin-induced assembly of the esterase within cells, showing increasing chemiluminescence with increasing rapamycin concentration and incubation time (Figure 12B).…”

Section: Biological Measurements and Applicationsmentioning

confidence: 99%