2025 AIChE Annual Meeting

(182u) Expanding ER Sequestration and Screening for Enzyme Engineering through Nanobody or Spy Tag-Mediated ER Retention.

Authors

David Arceo Zamora, University of Florida
Sarah Murphy, University of Florida
Carl Denard, University of Texas at Austin
Yeast ER sequestration and screening is now a well-established strategy for enzyme reprogramming. Specifically, the Yeast ER sequestration system (YESS) demonstrates that one can leverage an ER retention mechanism to evolve proteases with improved selectivity1. Generally, there are two main units – the substrate and protease unit. The substrate unit is coupled to an ER signal peptide (sp) and Aga2p mating adhesion receptor on the N-terminus and ER retention sequence (ERS) on the C-terminus with epitope tags on each side of the substrate to monitor substrate cleavage. The protease unit also contains a sp and ERS. Now, upon protease and substrate expression, both are localized and retained in the ER by ERS interactions with the ER membrane receptor Erd22. As a result, this system enables proximity-based catalysis, allowing the protease to cleave the substrate, provided it possesses the appropriate specificity and activity. Subsequently, only the substrate is transported to the cell surface, displayed on the surface, and can be sorted using flow cytometry by labeling surface-displayed substrate epitopes with fluorescently labeled anti-epitope antibodies. YESS and its counterparts, such as YESS 2.0 and PERRC, demonstrate that ER retention is a powerful strategy for protease evolution, specifically for a model enzyme such as Tobacco Etch Virus protease (TEVp)1,3,4. However, a key challenge can arise when applying these systems to therapeutically relevant or structurally complex proteins, as some may fail to properly fold or remain active in these configurations. As previously described, ER retention depends on the ERS, and currently, it must be attached to the C-terminus of the protein. This limits the orientation in which you can tag a protein for retention, and C-terminal labeling may interfere with specific protein folding, structure, and function. This is especially significant for proteins with critical C-terminal elements. However, it is not feasible to simply attach the ERS to the N-terminus of the protein5.

Here, we present a modified approach to facilitate ER retention, allowing for modular ERS orientation using an intermediate mediator between the protease and the ERS. In this system, there are now 3 transcriptional units that are controlled under three orthogonal inducible promoters: 1) Under a galactose inducible promoter, the substrate unit – which is configured as previously described 2) Under an aldosterone inducible promoter, a modified protease unit 3) Under a β-estradiol promoter, a newly added mediator unit. Now, the modified protease unit contains a sp followed by a recognition tag (RecTag) that can be labeled on the protease's N- or C-terminus. The newly added mediator unit contains a sp on the N-terminus of the mediator and the ERS on the C-terminus. In this configuration, the ERS is indirectly linked to protease retention. The logic of the system is as follows: 1) the mediator unit is expressed, translocated in the ER, and is retained through interactions between ERS-Erd2 2) the protease and substrate unit can be induced and retained in the ER. The substrate through direct ERS-Erd2 retention and the protease through RecTag-mediator-ERS-Erd2 interaction. As a result, proximity-based catalysis between protease and substrate is enabled. 3) Lastly, the phenotype can be measured using flow cytometry. For this system, we leverage two RecTag-mediator pairs: AlfaTag-Nanobody Alfa (NbAlfa) and Spytag-Spycatcher6,7. We will demonstrate that these pairs can mediate ER retention and modularly retain proteases at their N- and C-termini. The newfound ability of N-terminal labeling may alleviate interference that may have been observed from previous C-terminal ERS systems by preserving the C-terminal region. Accordingly, we will show that this modified system can be used to screen and evolve proteins that were inactive in previous systems. This approach expands the toolbox for Yeast ER sequestration and broadens the scope of proteins that can be screened using these ER sequestration systems, enabling the discovery and engineering of previously uncharacterized proteins with enhanced functionality.

References:

(1) Yi, L.; Gebhard, M. C.; Li, Q.; Taft, J. M.; Georgiou, G.; Iverson, B. L. Engineering of TEV Protease Variants by Yeast ER Sequestration Screening (YESS) of Combinatorial Libraries. Proc. Natl. Acad. Sci. 2013, 110 (18), 7229–7234. https://doi.org/10.1073/pnas.1215994110.

(2) Semenza, J. C.; Hardwick, K. G.; Dean, N.; Pelham, H. R. B. ERD2, a Yeast Gene Required for the Receptor-Mediated Retrieval of Luminal ER Proteins from the Secretory Pathway. Cell 1990, 61 (7), 1349–1357. https://doi.org/10.1016/0092-8674(90)90698-E.

(3) Denard, C. A.; Paresi, C.; Yaghi, R.; McGinnis, N.; Bennett, Z.; Yi, L.; Georgiou, G.; Iverson, B. L. YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants. ACS Synth. Biol. 2021, 10 (1), 63–71. https://doi.org/10.1021/acssynbio.0c00452.

(4) Nelson, S.; Gaza, J.; Ajayebi, S.; Masse, R.; Pho, R.; Scutero, C.; Martinusen, S.; Long, L.; Menezes, A.; Perez, A.; Denard, C. PERRC: Protease Engineering with Reactant Residence Time Control. March 4, 2025. https://doi.org/10.1101/2025.03.02.641063.

(5) Mei, M.; Zhai, C.; Li, X.; Zhou, Y.; Peng, W.; Ma, L.; Wang, Q.; Iverson, B. L.; Zhang, G.; Yi, L. Characterization of Aromatic Residue–Controlled Protein Retention in the Endoplasmic Reticulum of Saccharomyces Cerevisiae. J. Biol. Chem. 2017, 292 (50), 20707–20719. https://doi.org/10.1074/jbc.M117.812107.

(6) Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M. Peptide Tag Forming a Rapid Covalent Bond to a Protein, through Engineering a Bacterial Adhesin. Proc. Natl. Acad. Sci. 2012, 109 (12). https://doi.org/10.1073/pnas.1115485109.

(7) Götzke, H.; Kilisch, M.; Martínez-Carranza, M.; Sograte-Idrissi, S.; Rajavel, A.; Schlichthaerle, T.; Engels, N.; Jungmann, R.; Stenmark, P.; Opazo, F.; Frey, S. The ALFA-Tag Is a Highly Versatile Tool for Nanobody-Based Bioscience Applications. Nat. Commun. 2019, 10 (1), 4403. https://doi.org/10.1038/s41467-019-12301-7.