Box 2 Use of fusion proteins for pull-down assays in the study of signalling protein domain interactions
Individual domains often retain their function when isolated from their parent protein, and they can be genetically engineered to be fused with other proteins/peptides. Recombinant fusion proteins consist of two proteins: (a) a protein or peptide sequence used as a tag to facilitate protein isolation; and (b) the ‘bait’ protein, used as a means of indirectly ‘pulling down’ interacting proteins. One very useful example of this technique is the use of glutathione S -transferase (GST) fusion proteins; they consist of GST (tag) fused to a protein or part of a protein of interest (bait).
Using recombinant DNA technology, the DNA encoding the domain of interest is inserted into a plasmid vector just downstream of, and in the same translation reading frame (ORF) as the gene for GST. Under optimized conditions, certain strains of bacteria are induced to take up the plasmid and grown in selection media. Expression of the fusion protein is then chemically induced in transformed bacteria, which are subsequently lysed in a detergent solution. GST is used as the fusion partner because it binds glutathione, a property that can be exploited to purify the fusion protein by affinity chromatography. Free glutathione can be used to elute the fusion protein from the column. The GST can then be cleaved from the protein being investigated, if not further required. This gentle technique produces fusion protein of sufficient quantity and quality for use in, for example, binding assays and enzyme activity assays. Figures 16 and 17 show one example of its use.