Transforming a (beta/alpha)8--barrel enzyme into a split-protein sensor through directed evolution.
Split-protein sensors have become an important tool for the analysis 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 (beta/alpha)(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.
Tafelmeyer
Petra
P
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Johnsson
Nils
N
Johnsson
Kai
K
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D015374:Biosensing Techniques / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017384:Sequence Deletion; D014364:Tryptophan / Q000737:chemistry
D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase