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Limitation of Yeast Two-hybrid
- Dec 05, 2018 -

Yeast two-hybrid system is an effective and rapid method to analyze protein-protein interaction. two-hybrid system analysis of protein interactions located in the nucleus, and many protein interactions rely on post-translational processing such as glycosylation, disulfide bond formation, and so on, these reactions can not be carried out in the nucleus.In addition, the correct folding and function of some proteins depend on the assistance of other non-yeast proteins, which limits the research of some extracellular proteins and cell membrane receptor proteins.An important problem for the yeast two-hybrid system is "false positives".Because some proteins have transcriptional activation function or play transcriptional activation role when expressed in yeast, DNA binding domain hybrid proteins can activate transcription without specific domain activation.In addition, the surface of some proteins contains low affinity regions for a variety of proteins, which can form stable complexes with other proteins, thus causing the expression of reporter genes and producing "false positive" results.

Many researchers have improved and developed dual hybrid systems.For example, the use of false positive display analysis and double screening system to reduce the occurrence of "false positive";Develop mammalian dual hybridization systems to better study protein interactions.(2) there are two different reporter genes (commonly used for lacZ and HIS3) in the dual-screening system with the following advantages.The essay enhances the screening ability through nutritional screening, especially for screening with a large reservoir capacity and fewer selected proteins.

Mammals Ⅷ double hybrid system is a kind of gene level based on the reconstruction of transcription factor function analysis method in the body.In this system, one protein of interest binds to Gal4--DNA domain to form fusion protein, and another protein is expressed by fusion protein with the activation domain of herpes simplex virus VP16 protein.The vector expressing these fusion proteins was co-transfected with a reporter vector (CAT) into mammalian cell lines.The plasmid contains a cat gene downstream of Gal4 binding site.If the two fusion proteins interact, the cat reporter gene expression level significantly increases.The interaction between P53 protein and large T antigen was confirmed by this system.The results were obtained within 48h after transfection.And mammalian cells are better able to mimic protein-protein interactions in the body.Therefore, the mammalian two-hybrid system can be used as an auxiliary means of yeast two-hybrid system.

 

Dec.05, 2018

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