Expression, purification, characterization and crystallization of Panax quinquefolius ginsenoside glycosyltransferase Pq3-O-UGT2

• Published in: Protein expression and purification 2024-04, Vol.216, p.106430-106430, Article 106430
• Main Authors: Ji, Qiushuang, Liu, Yirong, Chen, Cheng, Zhang, Huanyu, Wang, Juan, Mei, Kunrong
• Format: Article
• Language: English
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2024.106430

Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant Pq3-O-UGT2 was purified through Ni-NTA affinity purification, a two-step ion exchange chromatography, and subsequently size-exclusion chromatography (SEC). Notably, the purified Pq3-O-UGT2 showed substantial activity toward Rh2 and F2, catalyzing the formation of Rg3 and Rd, respectively. This activity was discernible within a pH range of 4.0-9.0 and temperature range of 30-55 °C, with optimal conditions observed at pH 7.0-8.0 and 37 °C. The catalytic efficiency of Pq3-O-UGT2 toward Rh2 and F2 was 31.43 s  mΜ and 169.31 s  mΜ , respectively. We further crystalized Pq3-O-UGT2 in both its apo form and co-crystalized forms with UDPG, Rh2 and F2, respectively. High-quality crystals were obtained and X-ray diffraction data was collected for all co-crystalized samples. Analysis of the diffraction data revealed that the crystal of Pq3-O-UGT2 co-crystalized with UDP-Glc belonged to space group P1, while the other two crystals belonged to space group P2 2 2 . Together, this study has laid a robust foundation for subsequent structural analysis of Pq3-O-UGT2.