# Strategies for Post-Translational Control of Protein Expression and Their Applications
## 4 Conditional Control of Protein Stability Using Genetic Manipulation
### Excisable Degrons  
Most of the methods that use the degrons mentioned above, with the exception of LiPD and DiPD, require permanent fusion with degrons at the N or C terminus of the POI. The terminal region of the protein often encodes an important sequence that is indispensable for proper function, such as post-translational modification or interaction with other proteins. This prompted researchers to develop excisable degrons that allow the release of target proteins from degrons. [PERSON] et al. developed the split Ub for rescue of function (SURF) system [75], in which cells express a fusion protein of FKBP12 with the Ub N-terminal fragment and a fusion protein of three copies of the FRB mutant W2101F (used as a degron) to the Ub C-terminal fragment, followed by the POI (Figure 6a). Rapamycin induces the dimerization of FKBP12 and FRB mutant, to allow Ub fragment complementation. The Ub domain is cleaved by cellular DUBs, releasing the POI from the FRB mutant. The SURF system allows the conditional control of protein stability in native from, but requires dimerization of two elements in conjunction with transduction of multiple genes.  
Figure 6: Excisable degrons. Concepts of (**a**) SURF, (**b**) SMASH, and (**c**) LIBRON.  
[PERSON] et al. developed small-molecule-assisted shutoff (SMASH), which is a single-ligand/single-domain technique [76]. In this methodology, the POI is fused with the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease domain, followed by hydrophobic sequences of HCV NS4A, which are employed as a degron (Figure 6b). In the ligand-free state, an untagged POI is generated through cleavage via internal HCV NS3 protease activity; in turn, the cleavage can be blocked by the addition of an HCV protease inhibitor, either cluprevir (CLV) or asunaprevir (ASV), thus inducing degradation of the fusion proteins. Despite being an elegant strategy, this technique does not compensate for the release of the completely native form of the protein, as amino acids of the protease-recognition site remain in the POI after the removal of the degron.  
Recently, our group published a method to conditionally control protein stability in native or near-native form. This method combines the destabilizing domain (DD) technology with the enzymatic action of the abundant cellular DUBs, thus allowing the liberation of the POI from the degron tag, which is termed a liberation-prone degron (LIBRON) (Figure 6c) [77]. We introduced a lysine-free Ub variant (referred to as Ub*) as a cleavage tag between the C terminus of the DD and the N terminus of the POI. The rationale behind the LIBRON system consists of tuning the cleavage speed of the DUB enzyme as an Ub with a wild-type C terminus sequence is cotranslationally processed by an abundant cellular enzyme. We found that G75S/C mutations yielded moderate resistance to DUB cleavage, which compensated for the control of protein degradation. When the Ub*G75S/C mutants are inserted as cleavage tags between the DD and the POI, the entire fusion protein is degraded because of the instability of the DD when expressed in mammalian cells. The addition of a stabilizing ligand bound to the DD rescues it from degradation, followed by the release of the POI from C-terminus site of the DD-Ub*G75S/C tag. The LIBRON system is useful for the conditional regulation of proteins that require unmodified N-termini for their functionality.