A sweet protein monellin as a non-antibody scaffold for synthetic binding proteins

Synthetic binding proteins that have the ability to bind with molecules can be generated using various protein domains as non-antibody scaffolds. These designer proteins have been used widely in research studies, as their properties overcome the disadvantages of using antibodies. Here, we describe the first application of a phage display to generate synthetic binding proteins using a sweet protein, monellin, as a non-antibody scaffold. Single-chain monellin (scMonellin), in which two polypeptide chains of natural monellin are connected by a short linker, has two loops on one side of the molecule. We constructed phage display libraries of scMonellin, in which the amino acid sequence of the two loops is diversified. To validate the performance of these libraries, we sorted them against the folding mutant of the green fluorescent protein variant (GFPuv) and yeast small ubiquitin-related modifier. We successfully obtained scMonellin variants exhibiting moderate but significant affinities for these target proteins. Crystal structures of one of the GFPuv-binding variants in complex with GFPuv revealed that the two diversified loops were involved in target recognition. scMonellin, therefore, represents a promising non-antibody scaffold in the design and generation of synthetic binding proteins. We termed the scMonellin-derived synthetic binding proteins “SWEEPins.”

Expression of Brazzein, a Small Sweet-Tasting Protein in Saccharomyces cerevisiae: An Introduction for Production of Sweet Yeasts

Background: The replacement of carbohydrate sweeteners with protein sweeteners from plants has attracted the interest of researchers because these proteins don’t trigger the insulin response and are more nutritive for consumption in food. Brazzein (Braz) is a small and heat- stable sweet protein that has been originally derived from African plant Pentadiplandra brazzeana. In the present work the solubility, sweetness and yield of recombinant forms of Braz in two expression hosts, E. coli and S. cerevisiae were comprised. Methods: The codon-optimized gene of Braz was cloned in expression vectors pET28a and pET41a and GPD. The resulted vectors pET28a-Braz and pEt41a-Braz were transformed into Escherichia coli strain Rosetta (DE3) and the vector GPD-Braz was transformd to S. cerevisiae. The expression of Braz in different systems was analyzed by SDS-PAGE and western blotting. Results: The results verified the heterologous expression of Braz in S. cerevisiae carrying GPDBraz. Also the expression of Braz as carboxy-terminal extensions of His-tag and Glutathione-STransferase (GST) were verified in transgenic E. coli containing pET28a-Braz and pET41a-Braz, respectively. Conclusion: Although the yield of GST-Braz was higher than His-Braz and Braz expressed in S. cerevisiae, but the higher solubility, sweetness, safety (GRAS) are important advantages of the use of S. cerevisiae as expression host for production of Braz. Therefore the result of present work opens new insights for providing the new sweet yeasts that can be used as food additives.

Metabolic Effects of the Sweet Protein MNEI as a Sweetener in Drinking Water. A Pilot Study of a High Fat Dietary Regimen in a Rodent Model

Sweeteners have become integrating components of the typical western diet, in response to the spreading of sugar-related pathologies (diabetes, obesity and metabolic syndrome) that have stemmed from the adoption of unbalanced dietary habits. Sweet proteins are a relatively unstudied class of sweet compounds that could serve as innovative sweeteners, but their introduction on the food market has been delayed by some factors, among which is the lack of thorough metabolic and toxicological studies. We have tried to shed light on the potential of a sweet protein, MNEI, as a fructose substitute in beverages in a typical western diet, by studying the metabolic consequences of its consumption on a Wistar rat model of high fat diet-induced obesity. In particular, we investigated the lipid profile, insulin sensitivity and other indicators of metabolic syndrome. We also evaluated systemic inflammation and potential colon damage. MNEI consumption rescued the metabolic derangement elicited by the intake of fructose, namely insulin resistance, altered plasma lipid profile, colon inflammation and translocation of lipopolysaccharides from the gut lumen into the circulatory system. We concluded that MNEI could represent a valid alternative to fructose, particularly when concomitant metabolic disorders such as diabetes and/or glucose intolerance are present.

SWEET Transporters for the Nourishment of Embryonic Tissues during Maize Germination

In maize seed germination, the endosperm and the scutellum nourish the embryo axis. Here, we examined the mRNA relative amount of the SWEET protein family, which could be involved in sugar transport during germination since high [14-C]-glucose and mainly [14-C]-sucrose diffusional uptake were found in embryo tissues. We identified high levels of transcripts for SWEETs in the three phases of the germination process: ZmSWEET4c, ZmSWEET6b, ZmSWEET11, ZmSWEET13a, ZmSWEET13b, ZmSWEET14b and ZmSWEET15a, except at 0 h of imbibition where the abundance of each ZmSWEET was low. Despite the major sucrose (Suc) biosynthesis capacity of the scutellum and the high level of transcripts of the Suc symporter SUT1, Suc was not found to be accumulated; furthermore, in the embryo axis, Suc did not decrease but hexoses increased, suggesting an efficient Suc efflux from the scutellum to nourish the embryo axis. The influx of Glc into the scutellum could be mediated by SWEET4c to take up the large amount of transported sugars due to the late hydrolysis of starch. In addition, sugars regulated the mRNA amount of SWEETs at the embryo axis. These results suggest an important role for SWEETs in transporting Suc and hexoses between the scutellum and the embryo axis, and differences in SWEET transcripts between both tissues might occur because of the different sugar requirements and metabolism.

The Flexible Loop is a New Sweetness Determinant Site of the Sweet-Tasting Protein: Characterization of Novel Sweeter Mutants of the Single-Chain Monellin (MNEI)

The single-chain monellin (MNEI) displays same sweet potency as the natural monellin protein. To identify critical residues determining its sweetness, residues located at the loops region were selected for mutagenesis analysis. Mutations of positive-charge residues R31, R53, and R82 consistently led to obvious decrease of sweetness, whereas mutations of negative-charge residues resulted in variable sweet potency. Of note, the E50N mutant in the loop region linking the 2 natural chains showed significantly increased sweetness. Mutations of this residue to M or K led to similar effects, in accordance with the so-called wedge model for explanation of the sweet protein–receptor interaction. Homology modeling was carried out with the firstly reported crystal structure of sweet taste receptor (from medaka fish) as the template, and molecular docking and dynamics simulations suggested that flexible conformations of specific residues located in the loops region play essential roles for the interaction with the receptor and the sweetness of the protein. Moreover, obvious additive effects were found for the sweetness as 2 double-site mutants (E50N/Y65R and E2N/E50N) displayed increased sweetness than their single-site mutants. Our results revealed the flexible loop L23 linking the 2 natural chains as a novel sweetness determinant site of the sweet protein monellin and raised a series of new sweeter mutants, which could provide helpful guidance for molecular designing the sweet-tasting proteins.