Novel gnd_v2 Fusion Tag and Engineered TEV Protease Enable Efficient Production of Brazzein
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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 protein 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.
Abstract
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 protein 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.
참고문헌 (45)
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