Transforming a (beta/alpha)8-Barrel Enzyme into a Split-Protein Sensor through Directed Evolution

Kai Johnsson, Petra Tafelmeyer
2004
Article

Résumé

Split-protein sensors have become an important tool for the anal. of protein-protein interactions in living cells. We present here a combinatorial method for the generation of new split-protein sensors and demonstrate its application toward the (b/a)8-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. 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. This powerful selection complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening. [on SciFinder (R)]

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https://infoscience.epfl.ch/record/81367?ln=fr

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Publications associées (2)

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

Kai Johnsson, Petra Tafelmeyer

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)]

(Ecole Polytechnique Federale De Lausanne, Switz.).2005

Chargement

The invention concerns a combinatorial method for the generation of new split-protein sensors, and its application towards the (beta/alpha)8-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae is demonstrated. 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. This powerful selection thus complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening.

2005

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