Over-Expression, Detergent Extraction, Purification, and Preliminary Analysis of Recombinant Ferroportin from Various Vertebrate Species |

ABSTRACT:

Thispaper focuses on the over-expression, detergent-extraction and purification ofhuman, mouse, and zebrafish forms of ferroportin (Fpn) for use in variousbiophysical analyses and crystallization attempts. Prior to our studies,recombinant Fpn expression had been limited to small-scale eukaryotic systems.For example, the initial studies in which Fpn was identified and partiallycharacterized involved small-scale recombinant expression Fpn utilizing suchmethods as microinjection in Xenopus oocytes(Donovan et al. 2000; McKie et al. 2000) or zebrafish embryos(Donovan et al. 2000), or bytransient transfection in mammalian tissue culture lines, such as  Madin-Darby canine kidney cells (MDCK) (McKieet al. 2000), CaCo-2 cells(McKie et al. 2000), or HEK293Tcells (Abboud and Haile 2000). Fpn expression in these systems was evaluated bysensitive detection  techniques such  as  Western  blotting, direct  fluorescence  (in the  case  of Fpn-  GFP),  or immunofluorescence.    Subsequent Fpn studies used these same small-scale eukaryoticexpression methods (De  Domenico  et  al.  2005,  2006, 2007a,b,c;