Brazzein is an intensely sweet protein with high stability over a wide range of pH values and temperatures, due to its four disulfide bridges. Recombinant brazzein production through secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect disulfide formation, which is crucial for achieving the final protein structure and stability. Protein disulfide bond formation requires protein disulfide isomerase (PDI) and Ero1p. Here, we overexpressed KlPDI in K. lactis or treated the cells with dithiothreitol to overexpress KlERO1 and improve brazzein secretion. KlPDI and KlERO1 overexpression independently increased brazzein secretion in K. lactis by 1.7-2.2- and 1.3-1.6-fold, respectively. Simultaneous overexpression of KlPDI and KlERO1 accelerated des-pE1M-brazzein secretion by approximately 2.6-fold compared to the previous system. Moreover, intracellular misfolded/unfolded recombinant des-pE1M-brazzein was significantly decreased. In conclusion, increased KlPDI and KlERO1 expression favors brazzein secretion, suggesting that correct protein folding may be crucial to brazzein secretion in K. lactis.
Determining the in vivo gustatory thresholds of sweet-tasting molecules is more difficult and laborious than other sensory analyses. In this study, we refined the single-interval adjustment matrix (SIAM) yes-no task to produce threshold estimates accurately and efficiently. Experimental procedures were optimized such that tasters provided bias-free results and achieved maximum performance. We found that the refined SIAM yes-no task accurately estimated the threshold values of the sweet-tasting protein brazzein within the sensitive threshold range, which was confirmed using the two-alternative forced-choice task. Threshold values of various artificial sweeteners were also investigated using the SIAM yes-no task. Our results suggest that the refined SIAM yes-no task is advantageous for obtaining thresholds and comparing the biological activity of various sweet-tasting molecules.
Practical applicationsThe refined SIAM yes-no task suggested in this study would enable researchers to accurately obtain thresholds of various sweet-tasting molecules, in order to develop potential sugar substitutes and elucidate interactions with their corresponding sweet-taste receptors T1R2-T1R3 for sweet-taste mechanism studies.
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