Membrane Protein Inducible Overexpression Cell Line Development
Creative Biolabs has been a leader in the field of membrane protein and can provide you professional service for membrane protein stable cell line development. During all the phases, development plays a vital role in building the bridge from discovery to manufacturing for economic competitiveness.
The inducible method can be utilized to avoid the negative effects of constitutive membrane protein overexpression on cell development. In an inducible system, the expression can be switched on or off by changing an external factor, such as temperature or the addition of a chemical. As a result, during large-scale cell development, protein expression is restricted, allowing mammalian cell cultures to achieve the requisite high cell density. Expression is turned on for a short amount of time at this point, usually 24-72 hours before the cells are harvested. Comparative studies of constitutive and inducible expression of various membrane proteins have revealed that a tetracycline inducible expression system can produce 4- to 12-fold more membrane protein than its constitutive expression.
Fig.1. Inducible overexpression cell line. (Gomez-Martinez, 2013).
Tetracycline Inducible System
Researchers have adapted the Tetracycline repressor protein (TetR), taken from the E.coli tetracycline resistance operon, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system). The Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression.
Fig.2. Tetracycline inducible expression system.
DMSO Inducible System
In the bioprocessing sector, temperature shift cultivation is commonly used to improve recombinant protein production. Researchers discovered and characterized transcriptional regulatory elements that regulate cold inducible RNA-binding (CIRP) gene expression, demonstrating that they can be exploited to improve transgenic expression. Experiments found the basal transcriptional regulatory components of CIRP within 264 nucleotides upstream of the transcription start site, confirming the core promoter. The deletion of a region ranging from -264 to -64, which contained two potential CAAT-binding sites, resulted in the loss of promoter activity. In the region -452 to -264 of the transcription start site, a second promoter was discovered that could induce transcription independently of the core promoter.
Cold Inducible System
The differentiation of Friend erythroleukemia cells in vitro is a useful model for studying erythroid differentiation. Treatment of these cells in culture with DMSO induces an increase in heme biosynthesis pathway enzymes, globin mRNA, and cell membrane transferrin receptors, resulting in the synthesis and storage of hemoglobin in the cells.
Utilizing the highest qualified platforms and technicians, Creative Biolabs is quite experienced in cell line development. We provide flexible options for you. Any requirements, please feel free to contact us for further communication about your project.
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Reference
- Gomez-Martinez, M., et al. Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression. Journal of Visualized Experiments. 2013; 73: e50171.
