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 production. 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 extract. We describe the production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at their carboxyl-terminal ends. One of the constructs comprises the entire coding region for hTF (residues 1-679), while the other lacks the final three carboxyl-terminal amino acids. After insertion of the His-tagged hTFs into the pNUT vector, transfection into baby hamster kidney (BHK) cells, and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium. Average maximum expression levels 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 exchange column. The second step utilized a Poros metal chelate column preloaded with copper from which the His-tagged sample was eluted with a linear imidazole gradient. The His-tagged hTFs were characterized and compared to both 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 purification of these metal-binding proteins with minimal effects on many of their physical properties. The His-tagged hTFs share many features common to hTF, including reversible iron binding, reactivity with a monoclonal antibody, and presence as a monomer in solution.
Mason
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AB
Department of Biochemistry, University of Vermont, Burlington, Vermont 05405, USA. amason@salus.med.uvm.edu
He
Q Y
QY
Adams
T E
TE
Gumerov
D R
DR
Kaltashov
I A
IA
Nguyen
V
V
MacGillivray
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RT
D000345:Affinity Labels; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D006031:Glycosylation; D006639:Histidine; D006801:Humans; D007501:Iron / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D014162:Transfection; D014168:Transferrin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014168:Transferrin; D006639:Histidine; D007501:Iron