# Strategies for Post-Translational Control of Protein Expression and Their Applications
## 1 Introduction  
The manipulation of cellular functions is one of the major approaches in biological research and is supported by the control of biomolecules, including DNA, RNA, proteins, peptides, and bioactive small molecules. In particular, researchers often aim to control the activity of specific proteins in mammalian cells, to understand phenomena of interest. The observation of the cellular output and phenotypes in cases of selective enhancement or perturbation of the activity of the target protein enables the deduction of its function. For this purpose, a variety of methods [1] are now available that allow the manipulation of the cellular abundance, localization, conformational change, enzymatic activity, and post-translational modification of proteins, as well as their interaction with partners. Among them, controlling protein abundance is a robust approach compared with other methods because it directly ensures activity in cells. Altering the gene or transcript encoding the target protein is broadly used for the regulation of cellular protein abundance. The CRISPR/Cas9 system, which is one of the representative approaches in this setting, uses a Cas9 endonuclease and a single-guideRNA (sgRNA) with a complementary sequence to the target DNA [2; 3; 4]. The sgRNA guides the Cas9 to the target site in the genome, for cleavage. The CRISPR/Cas9 system allows for the precise and efficient deletion or insertion of the target DNA. The Cre/loxP system is another classical technique that also controls protein expression at the DNA level [5; 6; 7]. Cre recombinase specifically excises a DNA sequence flanked by two loxP sites. The Cre/loxP system allows the excision from the genome of a target DNA flanked by the loxP sites by expressing Cre proteins in a target tissue and at the desired time. Although the techniques of genetic depletion have greatly contributed to the advancement of biological studies, some concerns remain: first, it is not easy to apply them to the regulation of core-essential genes, which are indispensable for the viability of mammalian cells [8; 9]. Second, the living organism might adapt to the circumstances generated by the genetic depletion [10], which may mislead the interpretation of the phenotypic observations. Rather than controlling the target molecule at the DNA level, alternative approaches rely on the modulation of the gene of interest at the transcriptional level. The tetracycline (tet) system is a well-known tool for the transcriptional control of target protein expression that can be turned on or off using tet or its derivative, doxycycline (Dox) [11; 12; 13]. The tet system uses a fusion protein of the tetracycline ligand-binding domain to a sequence-specific transcription activation domain and a construct that contains a target gene under the control of a minimal promoter sequence combined with tet operator sequences. Another example of transcriptional control is the knockdown method based on RNA interference (RNAi), which is a regulatory mechanism present in most eukaryotic cells. RNAi uses small double-stranded RNA (dsRNA) molecules to direct homology-dependent degradation of the target mRNA [14; 15]. Although these techniques have been widely used as gold standards for the modulation of target proteins, these approaches face a fundamental limitation, i.e., waiting for the natural clearance of the target proteins from the cell [16]. In the past few decades, researchers have developed technologies that regulate protein expression at the post-translational level. Those technologies enable the fast clearance or stabilization of the target protein, because the stability of the target protein is controlled directly. Most technologies used for post-translational control of protein expression take advantage of cellular proteolytic pathways. Eukaryotic cells have two major proteolytic pathways: the ubiquitin-proteasome pathway [17] and the lysosomal pathway [18].