Expression and identification of recombinant human alpha-galactosidase A in Chinese hamster ovary cells

Objective To construct a recombinant eukaryotic expression plasmid of alpha-galactosidase A(GLA) and express GLA in Chinese hamster ovary cells(CHO).Methods Human GLA cDNA was cloned from human liver cancer cells by RT-PCR and subcloned into mammalian expression systems pcDNA3.1/myc-His A.The recombinant plasmid was transfected into CHO cells and the positive clones were selected by G418.After the G418 resistant CHO cells were induced by sodium butyrate,GLA activity of the supernatant was determined.The recombinant GLA was purified by HisTrapTM FF and identified by Western blot.Results The mammalian expression vector pcDNA3.1/myc-His-GLA was constructed and transfected into CHO cells.A transfectant with high GLA expression level was obtained(the protein specific activity was 1935 U/mg).There was an unique band near 50×103(Mr) in the purified supernatant and was identified as GLA protein by Western blot.Conclusion The recombinant eukaryotic expression plasmid of GLA is constructed and recombinant human GLA with biological activity is obtained.The recombinant human GLA can be used for Fabry′s disease treatment in the future.