To facilitate intein-mediated EPL of yeast-displayed VLRs, VLRs were cloned into the pCTre vector and fused to the engineered 202-08 intein using previously described methods (21, 22). For surface display experiments, 50 ml of yeast displaying VLR-intein fusion were pelleted, washed twice with 50 mM HEPES (pH 7.2), and then incubated with 200 mM MESNA in 50 mM HEPES (pH 7.2) (900 μl). This procedure reduces VLRs off the yeast surface and triggers the intein to form an unstable thioester. Immediately following MESNA addition, 5 mM 2mer peptide containing N-terminal cys and styrene handle was added to the yeast slurry (final volume, 1000 μl) (22). The mixture was incubated for 45 min at room temperature, and then, the yeast was pelleted. The supernatant was recovered and incubated at room temperature with shaking for an additional 2 to 18 hours to facilitate EPL. Buffer exchange of the VLR solution against PBS using 10 kDa MWCO filters removed MESNA and excess EPL ligand. Last, the tetrazine-Cy5 was incubated with VLR-styrene to facilitate covalent C-terminal modification of VLR and create VLR-Cy5 fusions. For secretion of intein-VLR fusions, VLRs were instead cloned into the pRS316 vector and fused to the 202-08 intein and produced and purified as noted in “Yeast culture” section above. Next, 200 mM MESNA was added to purified VLR-intein, followed by the addition of 5 mM Cys-PEG3-azide to produce VLR-azide fusions for liposome attachment. The mixture was incubated overnight at room temperature with gentle shaking. MESNA and excess EPL ligand were removed before use by buffer exchange with 10 kDa MWCO filters as described above. Western blots were used to characterize VLR-azide production. VLR samples were resolved on 4 to 12% bis-tris acrylamide and transferred to nitrocellulose using manufactures’ protocols. Blots were probed using anti-FLAG antibody (M1 murine monoclonal, Sigma) to detect the N-terminal FLAG tag.

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