(188bv) Facilitating Protease Engineering Using Golden Gate (GG) Assembly

Currently there are only 12 protease therapies on the market, and many new proteases and next generation proteases are in clinical development. Most current protease therapies are for treating cardiovascular disease, but there is promise for using them in treating inflammation, autoimmune diseases and cancer. However, in order to introduce new proteases to the market, they first need to engineered to be highly specific towards their in vivo targets. In our lab, we have developed the yeast endoplasmic reticulum sequestration system (YESS), a novel platform to engineer and profile protease specificity. YESS establishes contact between a protease and an ER-transiting protease substrate cassette destined for surface display. Any cleavage of the substrate by the engineered protease can be detected via fluorescence-activatedcell sorting (FACS) using fluorescently labeled antibodies that label epitope tags that flank the peptide substrate.

Here, we have used Golden Gate assembly to rapidly assemble YESS plasmids cheaply and with high efficiency. We restructured the YESS platform to allow a fast, modular, and efficient assembly of genetic elements that control transcription, translation, spatial sequestration and reporter tags. This seamless design-build-test cycle greatly speeds up our engineering and screening efforts. Using a combinatorial approach, Modular-YESS coupled with FACS quickly interrogates and modulates protease activity across multiple variables. We show examples for several proteases, including TEV protease, human elastases, human tissue kallikreins, and several metalloproteases, among others. Lastly, we show that modular YESS helps overcome engineering bottlenecks as it allows us to engineer a fast orthogonal TEV protease which prefers histidine at the P1 position. We believe Modular YESS is highly valuable to further advance recombinant protease therapeutics.