Analysis of the Cofactor Interaction

Two widely employed methods of studying receptor-cofactor interactions are the GST-pull-down and the mammalian 2-hybrid procedures. The GST-pull-down is an in vitro assay, whereas the mammalian 2-hybrid is performed in vivo. Both methods have advantages and limitations. The GST-pull-down protocol permits study of protein-protein interactions in a relatively pure and well-characterized system. Unfortunately expression of GST-fusion proteins much larger than 80,000 in molecular weight is often problematic due to poor yields and proteolytic degradation in the E. coli host. Use of a protease-impaired E. coli strain such as BL-21 can help minimize, but cannot fully eliminate this degradation problem. In the case of the mammalian 2-hybrid assay, protein-protein interactions can be characterized in an in vivo context that is likely to more accurately reflect normal physiology. However, the 2-hybrid interaction assay, of necessity, includes all the components of the cell; as a consequence, indirect interactions, as well as direct ones, may score by this procedure (see Note 10). It should be noted that both the GST-pull-down and 2-hybrid methods analyze protein-protein interactions in the absence of an appropriate DNA binding site. Therefore, we also describe a biotin/DNA pulldown assay that permits the characterization of the receptor-cofactor interac tion in the context of DNA binding. In this procedure, the receptor-cofactor complexes are assembled on a biotin-tagged DNA containing a suitable TRE. The protein/DNA complexes are then isolated using streptavidin-conjugated agarose, and the protein components of the complex are resolved by SDS-PAGE and visualized by radiolabeling or immunoblotting. This method also has the advantage of permitting the use of full-length native polypeptides.

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