Expression, purification, and characterization of recombinant nonglycosylated human serum transferrin containing a C-terminal hexahistidine tag

Attachment of a hexa-His tag is a common strategy in recombinant protein pr oduction. The use of such a tag greatly simplifies the purification of the protein from the complex mixture of other proteins in the media or cell ext ract. We describe the production of two recombinant nonglycosylated human s erum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa -His tag at their carboxyl-terminal ends. One of the constructs comprises t he entire coding region for hTF (residues 1-679), while the other lacks the final three carboxyl-terminal amino acids. After insertion of the His-tagg ed hTFs into the pNUT vector, transfection into baby hamster kidney (BHK) c ells, and selection with methotrexate, the secreted recombinant proteins we re isolated from the tissue culture medium. Average maximum expression leve ls of the His-tagged hTFs were about 40 mg/L compared to an average maximum of 50 mg/L for hTF-NG. The first step of purification involved an anion ex change column. The second step utilized a Poros metal chelate column preloa ded with copper from which the His-tagged sample was eluted with a linear i midazole gradient. The His-tagged hTFs were characterized and compared to b oth recombinant hTF-NG and glycosylated hTF from human serum. The identity of each of the His-tagged hTFs constructs was verified by electrospray mass spectroscopy. In summary, the His-tagged hTF constructs simplify the purif ication of these metal-binding proteins with minimal effects on many of the ir physical properties. The Histagged hTFs share many features common to hT F, including reversible iron binding, reactivity with a monoclonal antibody , and presence as a monomer in solution. (C) 2001 Academic Press.