sdAb Selection by Mammalian Two-Hybrid System
A mammalian two-hybrid system is a high-efficient tool for protein-protein interactions research. Besides, it is also a wonderful technology for functional sdAbs selection. Here, Creative Biolabs describes the selection of functional single domain antibody fragments for interfering with protein-protein interactions inside cells.
Introduction of Mammalian Two-Hybrid System
Protein-protein interactions (PPIs) is one of the most important mechanisms for activity regulation of mammalian cells. PPIs allow a fast cellular mechanism to respond to extracellular stimuli. As protein modification and regulation both involve PPIs, PPI characterization is essential to understand the mechanism of disease. In recent years, PPIs have been served as promising targets for different diseases.
The mammalian two-hybrid system is a powerful tool to identify and characterize protein-protein interactions in transiently transfected mammalian cells. Based on the modular domains found in some transcription factors, a completed system can be constructed for PPIs. Just similar to the yeast two-hybrid system, the mammalian two-hybrid system relies on the fusion of the DNA-binding domain (DBD) and the transcription activation domain (TAD) with the cDNAs of interest. The simian virus 40 (SV40) promoter involves the expression of two chimeric proteins. The first bait hybrid can be created by fusing the Gal4 DBD to mouse p53 and the second hybrid protein is constructed by fusing the VP16 activation domain to SV40 T antigen. Besides, the reporter gene is chloramphenicol acetyltransferase from cat controlled by five copies of the Gal4 binding sites upstream of the E1b minimal promoter.
Fig.1 Conventional mammalian two-hybrid assay.
Features of Mammalian Two-Hybrid System
- Rapid and quantitative
- Protein interactions in the mammalian two-hybrid system may better mimic actual in vivo environment
- Both nuclear and non-nuclear proteins can be detected
- Less suitable for cDNA library screenings
Mammalian Two-Hybrid System for sdAb Selection
Compared with conventional antibodies and antibody fragments, single domain antibodies (sdAbs) present great advantages in diagnostic and therapeutic applications. Once the target-specific sdAbs are obtained, the mammalian two-hybrid system can further be used to test their ability to bind the targets in mammalian cells and the biological functions of affection through interfering with their PPIs. Here, a triplex mammalian two-hybrid vector, which contains a dual promoter plasmid (Gal4 DBD & VP16 TAD), is used. And then the vector can be co-transfected with a plasmid expressing the candidate sdAbs and their effects are monitored by reporter activity. The main experiment steps contain 1). Plasmid construction; 2). Transfection into CHO reporter cell lines; 3). Luciferase reporter assay.
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