Method for identification of fragmentation sites in a reporter protein in the development of split protein sensors

A combinatorial method for the generation of new split-protein sensors that identifies sites at which a functional domain can be split to give two moieties that can be reconstituted to give a reporter or selectable activity is described. The method is demonstrated with the gene Trp1 N-(5'-phosphoribosyl)-anthranilate isomerase from Saccharomyces cerevisiae, which is (b/a)8-barrel enzyme. The resulting protein is described as a split-Trp sensor. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. Selection for tryptophan prototrophy is rapid and effective and can be used for high-throughput interaction screening. A series of deletion derivs. of the region of the gene encoding the catalytic domain of the protein was constructed by DNase I digestion of restriction fragments. These were combined with sequences encoding leucine zippers and tested pairwise for their ability to lift a tryptophan auxotrophy in yeast. Use of the split-Trp protein sensor to identify the interaction of the SEC62 and SEC63 gene products in the cell membrane of S. cerevisiae is demonstrated. [on SciFinder (R)]


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