Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin， with increased stability， were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system， reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produc... More
Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin， with increased stability， were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system， reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and the mutant clones were ranked based on fluorescence intensity. Mutants with increased affinity between the fragments， and subsequently increased stability， caused increased fluorescence intensity of split GFP. Single chain monellin variants of the top-ranked mutants for each chain， S76Y in the A-chain and W3C + R39G in the B-chain and all combinations thereof， were expressed and the increase in stability was verified by temperature denaturation studies using circular dichroism spectroscopy. Functionality studies showed that mutant S76Y has retained sweetness and has potential use within the food industry.
