Abstract:The gene encoding Echinococcus granulosus glyceraldehyde 3-phosphate dehydrogenase (EgGAPDH) and the structure and function of the encoded protein using bioinformatics were predicted in this study. And the EgGAPDH protein in E.coli prokaryotic expression system was cloned and expressed and assessed the enzyme activity of the protein in vitro. According to the predicted results such as physical and chemical properties, conserved domains, phylogenetic tree, and the secondary structure and 3D structure, the EgGAPDH gene was subcloned into high-level expressed vector pET30a(+), and the recombinant plasmid was subsequently transformed into competent E.coli BL21(DE3). The recombinant protein was expressed and purified. And its catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate(3-GAP). Results showed that the EgGAPDH cDNA, encoding a 336 amino acid protein with a predicted molecular weight of 36 128.3 Da and PI value of 8.44, was composed of 1 011 bp. The EgGAPDH protein was composed of 6 kinds of functional sites and 2 typical GAPDH family functional domains. The recombinant plasmid pET30a(+)-EgGAPDH was constructed and the EgGAPDH protein was effectively expressed in supernatant of the lysis. The enzyme activity assay revealed that the purified EgGAPDH had a significant enzyme activity in vitro. In conclusion, the EgGAPDH gene can express in prokaryotic expression system efficiently with definite enzyme activity and it provides a significant reference for further exploration of its function in glucose metabolism.
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