Identification of sweet active site of sweet protein, thaumatin

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Thaumatin was purified from the crude sample with ion-exchange chromatography and G-75 Sephadex chromatography. The obtained thaumatin showed a single band on PAGE and SDS-PAGE.The arginine residue of thaumatin was chemically modified with 1,2 cyclohexandione, the lysine residue with pyridoxal phosphate, and the tyrosine residue with sodium iode. From these experiments it has been clarified that one or two residues of each residue are realted to express sweet activity of thaumatin. In order to identify these residues the modified thaumatin was digested with pepsin and fractionated by HPLC.The peptide fragment containing the modified amino acid residue was obtained and applied to protein sequencer. To identify the active site of thaumatin, the sweet reception mechanism should be known. Then, isolation of receptor taste cells and thaumatin-receptor protein has been tried. Effects of pI on the sweet taste expression have been also examined