Data-driven protease engineering by DNA-recording and epistasis-aware machine learning
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Author / Producer
Date
2025
Publication Type
Journal Article
ETH Bibliography
yes
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Abstract
Protein engineering has recently seen tremendous transformation due to machine learning (ML) tools that predict structure from sequence at unprecedented precision. Predicting catalytic activity, however, remains challenging, restricting our capabilities to design protein sequences with desired catalytic function in silico. This predicament is mainly rooted in a lack of experimental methods capable of recording sequence-activity data in quantities sufficient for data-intensive ML techniques, and the inefficiency of searches in the enormous sequence spaces inherent to proteins. Herein, we address both limitations in the context of engineering proteases with tailored substrate specificity. We introduce a DNA recorder for deep specificity profiling of proteases in Escherichia coli as we demonstrate testing 29,716 candidate proteases against up to 134 substrates in parallel. The resulting sequence-activity data on approximately 600,000 protease-substrate pairs does not only reveal key sequence determinants governing protease specificity, but allows to build a data-efficient deep learning model that accurately predicts protease sequences with desired on- and off-target activities. Moreover, we present epistasis-aware training set design as a generalizable strategy to streamline searches within enormous sequence spaces, which strongly increases model accuracy at given experimental efforts and is thus likely to have implications for protein engineering far beyond proteases.
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Publication status
published
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Book title
Journal / series
Volume
16 (1)
Pages / Article No.
5466
Publisher
Nature
Event
Edition / version
Methods
Software
Geographic location
Date collected
Date created
Subject
High-throughput screening; Machine learning; Next-generation sequencing; Protein design; Protein function predictions
Organisational unit
03602 - Panke, Sven / Panke, Sven
Notes
Funding
179521 - XenoImport - Towards a generic synthetic import system for Escherichia coli (SNF)
965135 - Synthetic Biology of mRNA (EC)
965135 - Synthetic Biology of mRNA (EC)