(F-201) Generation and evaluation of proteases with high substrate sequence specificity

Introduction:: Proteases are enzymes that cleave peptide bonds in their substrate proteins.¹ Sequence-specific proteases are useful for research applications such as recombinant protein purification and therapeutics because they can be used to cleave specific peptide sequences and to target disease-associated proteins without off-target effects.^2,3 The Potyviridae plant virus family has specific proteases whose putative target sequences have been determined.⁴ We have engineered a number of these proteases to work in animal cells and tested their cleavage efficiency in vivo and in vitro. With data generated from our experiments, we will develop a machine learning model that can predict protease-substrate pairs de novo. Predicted protease sequences will be produced and tested in vitro and in vivo, repeating the modeling process until we have created a library of orthogonal enzymes for a wide range of protein substrates.


Materials and Methods:: We previously developed a fluorescence-based assay to determine protease activity in vitro.⁵ Briefly, three plasmids, each encoding for (1) a candidate protease, (2) a substrate protein with its putative cleavage site and a split fluorescent protein sfCherry(1-10), and (3) the other split protein sfCherry(11) bound to histone 2b are transfected into human embryonic kidney cells (HEK293T). Cleavage-induced reconstitution of fluorescence is measured 24 hours after transfection (Figure 1).

The putative proteolytic site is also introduced in the prodomain sequence of Vg1, a secreted signaling protein important in mesendoderm formation. Successful cleavage of the cut site by the protease results in proper embryo development.⁶ mRNAs encoding the protease and protease-cleavable Vg1 are injected in single-cell zebrafish embryos, and proper development is scored at 30 hours post-injection.


Results, Conclusions, and Discussions:: We tested 31 protease-substrate combinations in vitro, 15 of which exhibited >10-fold cleavage-induced fluorescence (Figure 2). Vg1 cleavage experiments in zebrafish have also shown successful cleavage (and therefore proper development) in 4 protease-substrate pairs. Since the proteases are being tested in their non-native environment, i.e., in animal cells instead of in plant cells, we will further optimize their sequences through rational mutagenesis to improve solubility, for example.


By engineering Potyviral proteases to function in human and zebrafish cells, we have validated a library of highly specific proteases and determined their cleavage properties in animal cells. Future work will involve testing the protease library against targets other than their predicted substrates. The selectivity data will help train a machine learning model, which will be able to design protease-peptide pairs de novo.




Acknowledgements (Optional): :

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2. Frey, S., & Görlich, D. (2014). A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. Journal of Chromatography A, 1337, 95–105. https://doi.org/10.1016/j.chroma.2014.02.029

3. Yoo, T. H., Pogson, M., Iverson, B. L., & Georgiou, G. (2012). Directed Evolution of Highly Selective Proteases by Using a Novel FACS-Based Screen that Capitalizes on the p53 Regulator MDM2. ChemBioChem, 13(5), 649–653. https://doi.org/10.1002/cbic.201100718

4. Rodamilans, B., Shan, H., Pasin, F., & García, J. A. (2018). Plant Viral Proteases: Beyond the Role of Peptide Cutters. Frontiers in Plant Science, 9. https://doi.org/10.3389/fpls.2018.00666

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6. Dingal P. C. D. P., Carte, A. N., Montague, T. G., & Schier, A. F. (2021). Regulation of Vg1 biogenesis during mesendoderm induction. BioRxiv (Cold Spring Harbor Laboratory). https://doi.org/10.1101/2021.04.25.441333