Fig. 1. Design of Retained Endoplasmic Retained Secretion (RELEASE).

a Proteins of interest are fused to RELEASE and retained in the ER via the dilysine ER retention domain (purple diamond). Upon activation or expression of a protease such as TEVP (orange partial circle), the ER retention domain is removed (middle panel) and the protein of interest is transported through the constitutive secretory pathway. When reaching the Trans-Golgi Apparatus (right panel), the native furin endoprotease cleaves the linker region allowing the membrane-bound protein to be secreted. b RELEASE is a modular platform and can be modified to respond to different proteases and regulate different proteins of interest. c The C-terminal dilysine motif of RELEASE is required for SEAP retention and using a construct where the lysine residues were modified to alanines (KKXX-COOH → AAXX-COOH) increased SEAP secretion. There was no significant difference in signal between RELEASE and control cells without SEAP. d Co-expression of proteases such as TEVP (orange partial circle), or HCVP (yellow partial circle) with the respective RELEASE constructs increased SEAP secretion. e The cleavage efficiencies of HCVP RELEASE constructs were also affected by transmembrane selection and was improved by modifying the residues flanking the HCVP cut site with native linker proteins. Based on the steady-state solution of a kinetic model for proteolytic cleavage, we determined that the relation between RELEASE output and the amount of protease plasmids fits the Michaelis–Menten equation⁷. We therefore fit the titration curves using Michaelis–Menten equations and used K_m to represent the apparent cleavage efficiency of each design by its corresponding protease. A complete list of the calculated cleavage efficiencies for the different RELEASE constructs can be found in Supplementary Table 1. f Dual-input control of protein expression was achieved using traditional DOX-inducible systems and RELEASE. Using a DOX-inducible SEAP-RELEASE with a Rapalog-inducible split TEVP, we observed fine control of SEAP secretion over a large dynamic range. g By removing the furin cut site, RELEASE was amenable to control the surface display of proteins. h Increased surface display of membrane-bound GFP fused to RELEASE in response to TEVP (left panel) or HCVP (right panel). Each dot represents a biological replicate. Mean values were calculated from four (c–f) or three replicates (h). The error bars represent ±SEM. The results are representative of at least two independent experiments; significance was tested using an unpaired two-tailed Student’s t-test between the two indicated conditions for each experiment. For experiments with multiple conditions, a one-way ANOVA with a Tukey’s post-hoc comparison test was used to assess significance. ***p < 0.001, ****p < 0.0001.