Molecules (Nov 2022)
Molecular Cloning, Expression, and Functional Analysis of Glycosyltransferase (TbUGGT) Gene from <i>Trapa bispinosa</i> Roxb.
Abstract
Trapa bispinosa Roxb. is an economical crop for medicine and food. Its roots, stems, leaves, and pulp have medicinal applications, and its shell is rich in active ingredients and is considered to have a high medicinal value. One of the main functional components of the Trapa bispinosa Roxb. shell is 1-galloyl-beta-D-glucose (βG), which can be used in medical treatment and is also an essential substrate for synthesizing the anticancer drug beta-penta-o-Galloyl-glucosen (PGG). Furthermore, gallate 1-beta-glucosyltransferase (EC 2.4.1.136) has been found to catalyze gallic acid (GA) and uridine diphosphate glucose (UDPG) to synthesize βG. In our previous study, significant differences in βG content were observed in different tissues of Trapa bispinosa Roxb. In this study, Trapa bispinosa Roxb. was used to clone 1500 bp of the UGGT gene, which was named TbUGGT, to encode 499 amino acids. According to the specificity of the endogenous expression of foreign genes in Escherichia coli, the adaptation codon of the cloned original genes was optimized for improved expression. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbUGGT with squalene synthases from other plants. The TbUGGT gene was constructed into a PET-28a expression vector and then transferred into Escherichia coli Transsetta (DE3) for expression. The recombinant protein had a molecular weight of 55 kDa and was detected using SDS-PAGE. The proteins were purified using multiple fermentation cultures to simulate the intracellular environment, and a substrate was added for in vitro reaction. After the enzymatic reaction, the levels of βG in the product were analyzed using HPLC and LC-MS, indicating the catalytic activity of TbUGGT. The cloning and functional analysis of TbUGGT may lay the foundation for further study on the complete synthesis of βG in E. coli.
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