Bioinformatics Analysis,Expression,Purification And Detection Of Enzyme Activity Of The GAPDH Protein From Echinococcus Granulosus

Objective:To predict the gene encoding Echinococcus granulosus glyceraldehyde3-phosphate dehydrogenase(EgGAPDH) and the structure and function of the protein using bioinformatics. And to clone and express the EgGAPDH recomninant protein in E.coli prokaryotic expression system and measure the enzyme activity of the protein purified by Ni-NTA column in vitro.Methods:The full-length cDNA sequence of EgGAPDH and its protein sequence was analyzed using bioinformatics. After attaining the EgGAPDH gene from pBlue-EgGAPDH recomninant plasmid, we digested it with EcoR I and HandⅢ. Then the DNA fragment was collected, purified and subcloned into the proper prokaryotic expression vector pET30a(+) which was digested with the same restriction enzyme to construct the high-level expressed vector pET30a(+)-EgGAPDH. After identification of the sequence, the recombinant plasmid was transformed into competent E.coli BL21(DE3) and expressed in the presence of Isopropyl β-D-1-Thiogalactopyranoside(IPTG). The EgGAPDH protein was purified with Ni-NTA column, and then its catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate(3-GAP).Results:The EgGAPDH cDNA, encoding a336amino acid protein with a predicted molecular weight of36128.3Da and PI value of8.44, is composed of1011base pairs(bp). The predicted protein is composed of6kinds of function sites and two 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.Conclusion:pET30a(+)-EgGAPDH recombinant plasmid is constructed and expressed efficiently. The enzyme activity of EgGAPDH protein is identified, which provids a significant reference for further studying its function in glucose metabolism.