Saffron Stigma Glycosyl transferase (CsUGT) heterologous expression in E.coli

Saffron stigmas contain complicated groups of metabolites that are mostly available in glycosylated forms. The main case of diversity of secondary metabolites is a post modification on the main structure of these components like glycosylation, one of the participated key enzymes in glycosylation pathway is Glycosyl transferase(GTase). This study was conducted to investigate the isolation of Saffron stigmas GTase coding gene and subsequently assessment of heterologous expression of isolated gene in Bacteria. First of all, the desired gene was isolated by degenerate oligoes, sequencing results revealed that the gene is in 8631 bp length and associated to CsUGT protein family which secret into cytoplasm. Afterward to evaluate the enzyme activity firstly, the obtained sequence was sub-cloned in pThio expression vector by Gibson assembly technique, then the recombinant vector transformed into BL18-pGro7 which were able to express chaperon proteins with EL& ES subunits. The bacteria pellets obtained by ultrasound were suspended in PBS solution and then the soluble proteins were extracted by boiling method. Finally the protein electrophoresis result by SDS pages were showed that the recombinant protein of CsUGT were correctly expressed in bacteria. The gained results in this project could be helpful to identify the different types of CsUGT isomers and characterization of these enzyme functions.

Keywords:

Saffron , Bacteria , Heterologous expression