Abstract
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.
Highlights
Stability toward denaturation is of utmost importance for proteins to be used in a technical application, within the pharmaceutical industry or food industry
The split green fluorescent protein (GFP) system used here relies on non-covalent association of the NGFP-MNA and MNB-CGFP gene products
To design more stable mutants remains hard despite that key factors influencing protein stability, such as packing of the hydrophobic core, hydrogen bond formation, electrostatic interactions, conformational entropy and bond strain are rather well understood[43,44,45]
Summary
Stability toward denaturation is of utmost importance for proteins to be used in a technical application, within the pharmaceutical industry or food industry. We use an in vivo screening method based on split green fluorescent protein (GFP) and the thermodynamic linkage between fragment complementation and protein folding, first shown by Lindman et al.[7], to develop mutants of the sweet tasting protein monellin (MN) with high stability. Protein variants with increased affinity between the fragments (and subsequently increased stability) cause increased fluorescence intensity of split GFP by favoring reconstitution/folding of GFP and thereby chromophore maturation This approach enables selection of mutations in the protein of interest that favor the associated state more than the dissociated state as well as mutations that disfavor the dissociated state more than the associated state. We utilize the split GFP assay with the monellin subdomain fragments to obtain an even more stable variant of scMN, with retained sweetness, that can meet the stability requirements from the food industry
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