Kaila S. SRAI, Basharut A. SYED, Elif Sibel ASLAN, Kenneth N. WHITE, Robert W. EVANS

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

Hephaestin (Hp) is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originallyidentified 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 ironis normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin (Cp), a member of the family of multi copper ferroxidases (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 asecretory form tagged with green fluorescent protein (GFP), Hpsec-GFP. Plasmid expressing Hpsec-GFP was constructed and transfectedinto COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression andprovided a tag for purifying and analysing Hpsec-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-basedassays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteinsusing 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.

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