Protein Interactions in Intact Cells and Animals

Detecting Protein Interactions in Intact Cells and Animals 

A variety of techniques have been developed to investigate protein-protein interactions in cultured cells, including the two-hybrid system and protein-fragment complementation (PFC). The two-hybrid system is the most widely applied method to identify and characterize protein interactions. However, several features of PFC make it attractive as an approach for in vivo imaging of protein interactions in cells, and particularly, in live animals. Subsequently, we describe major features of these two methods and other strategies with potential utility for in vivo imaging. 

However, the two-hybrid method has limitations. Some types of proteins do not lend themselves to study by the two-hybrid method. For example, because production of signal in the two-hybrid method requires nuclear localization of the hybrid proteins, integral membrane proteins cannot be studied in their intact state. In addition, the time delay associated with transcriptional 

PFC assays depend on division of a monomeric reporter enzyme into two separate inactive components that can reconstitute function upon association [Fig. 12.2(a)]. When these reporter fragments are fused to interacting proteins, reporter activity is reconstituted upon association of the interacting proteins. PFC strategies based on several enzymes including /3-galactosidase, 

In live cells and in cell lysates, our optimized complementation system successfully reproduced published apparent values for rapamycin (25,27,30,33,34). 

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II. Detecting Protein Interactions in Intact Cells and Animals... 

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