Phage-assisted continuous evolution of proteases with altered substrate specificity
By M. Packer, H. Rees, David R. Liu. 2017
114 citations recorded by the source corpus.
Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications. Proteases are promising therapeutics to treat diseases such as hemophilia which are due to endogenous protease deficiency. Here the authors use phage-assisted continuous evolution to evolve a variant TEV protease with altered target peptide sequence specificities.
Subfields: Genetics, Immunology, Radiology, Nuclear Medicine and Imaging, Computational Biology & Bioinformatics
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Citations in the corpus
- Biological structure and function emerge from scaling unsupervised learning to 250 million protein sequences (2019, background): “…function (Romero & Arnold, 2009), inform protein design using a combination of activating mutants (Hu et al., 2018), and make rational substitutions to optimize protein properties such as substrate specificity (Packer et al., 2017), stability (Tan et al., 2014), and binding (Ricatti et al., 2019).”