Functional Expression of the Sweet-Tasting Protein Brazzein in Transgenic Tobacco Plants

The sweet-tasting protein, brazzein, has potential as a low-calorie sugar substitute owing to its high sweetness, stability, and water solubility. In this study, a synthetic brazzein gene was expressed in the tobacco plant, Nicotiana tabacum. Three types of expression cassettes containing the brazzein gene were constructed to examine the expression and purification efficiency of the brazzein: pBI-BZ1 containing a signal sequence and His-tag, pBI-BZ2 containing a signal sequence, and pBI-BZ3 containing only the brazzein gene. Brazzein, whose expression was confirmed by ELISA, was purified via ammonium sulfate precipitation, heat treatment, and CM-sepharose chromatography. The purity and conformational state of the brazzein were confirmed by SDS-PAGE, HPLC, and circular dichroism. The identity of the brazzein was confirmed by N-terminal amino acid analysis, ESI-MS/MS, and sweetness analysis. We successfully generated brazzein-overexpressing tobacco plants; thus, this method could be used to produce brazzein, which can be used as an alternative to currently produced sweeteners.