Novel gnd_v2 Fusion Tag and Engineered TEV Protease Enable Efficient Production of Brazzein

Protein solubility and purification challenges often hinder the large-scale production of valuable proteins like brazzein, a potent sweet protein with significant health benefits and commercial potential. This study introduces two novel tools to overcome protine expression and purification bottlenecks: a gnd_v2 fusion tag and an engineered Tobacco Etch Virus (TEV) protease. The gnd_v2 tag, derived from 6-phosphogluconate dehydrogenase, was engineered to improve the soluble expression of brazzein. This tag increased brazzein's solubility by four times compared to the wildtype gnd tag, marking a significant advancement in efficient brazzein production. To address the challenge of cleaving the fusion tag, we engineered a TEV protease variant with high efficiency, particularly at the glutamine residue at brazzein's P1' site - a known difficulty for wild-type TEV proteases. We achieved streamlined production of pure, functional brazzein by integrating this tailored protease cleavage with an ultrafiltration-based purification protocol. Notably, the purified brazzein demonstrated a sweetness potency approximately 2500 times that of sucrose, highlighting its potential as a high-intensity natural sweetener. While this study focused on brazzein, the gnd_v2 tag shows promise for enhancing the solubility of other challenging proteins. More broadly, this work presents a versatile toolset for the scalable production of diverse functional proteins, with significant implications for industrial applications in food and pharmaceutical domains.