Expression of soluble, active, fluorescently tagged hephaestin in COS and CHO cell lines

Authors : Elif Sibel ASLAN, Kenneth N WHİTE, Basharut A SYED, Kaila S SRAİ, Robert W EVANS

Pages : 393-405

View : 16 | Download : 8

Publication Date : 2020-12-01

Article Type : Research Paper

Abstract :Hephaestin insert ignore into journalissuearticles values(Hp); is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originally identified as the gene responsible for the phenotype of sex-linked anaemia in the sla mouse. The mutation in the sla protein causes accumulation of dietary iron in duodenal cells, causing severe microcytic hypochromic anaemia. Although mucosal uptake of dietary iron is normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin insert ignore into journalissuearticles values(Cp);, a member of the family of multi copper ferroxidases insert ignore into journalissuearticles values(MCFs); and possesses ferroxidase activity that facilitates iron release from the duodenum and load onto the serum iron transport protein transferrin. In the present study, attempts were made to produce biologically active recombinant mouse hephaestin as a secretory form tagged with green fluorescent protein insert ignore into journalissuearticles values(GFP);, Hpinsert ignore into journalissuearticles values(sec);-GFP. Plasmid expressing Hpinsert ignore into journalissuearticles values(sec);-GFP was constructed and transfected into COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression and provided a tag for purifying and analysing Hpinsert ignore into journalissuearticles values(sec);-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-based assays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteins using complementary fluorescent tags on target proteins that would facilitate the fluorescence resonance energy transfer measurements, for example with transferrin or colocalisation studies, and help to discover more about hephaestin works at the molecular level.
Keywords : Hephaestin, ceruloplasmin, ferroxidase, iron