D. Thanos, T. Maniatis
Methods in enzymology
Publisher Summary This chapter discusses the in vitro assembly of enhancer complexes. A large number of eukaryotic transcriptional enhancers and promoters have been characterized, and the transcriptional activator proteins that bind to these DNA sequences have been identified. Both enhancers and promoters have been shown to consist of multiple transcription-factor binding sites that result in the cooperative binding of the proteins in vitro and transcriptional synergy between them in vivo . Enhancers have also been shown to contain binding sites for so-called architectural proteins, enhancer components that have no intrinsic transcriptional activity, but are required for the assembly and function of enhancer complexes. The chapter describes a method for the rapid purification of recombinant proteins by metal-chelate affinity chromatography. The cDNA of the activator is engineered in such a way to encode six histidine residues (6His) at the amino or carboxyl terminus of the open reading frame. 6His-fused proteins are expressed in E. coli and can be purified to near homogeneity by a single-step affinity-chromatography procedure using a resin containing nickel ions (Ni 2+ ). A significant advantage of the metal-chelate affinity chromatography is that the histidine tail binds to the nickel resin even when the protein is denatured. The following protocol describes a procedure used for the purification of proteins under denaturing conditions. In this case, it is necessary to refold the protein and the methods used for renaturation are provided.