title: Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins.
abstract: Transferrin is a bilobal protein with the ability to bind iron in two binding sites situated at the bottom of a cleft in each lobe. We have previously described the production of recombinant non-glycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at the amino-terminus. Constructs in this background that contain strategic mutations to completely prevent iron binding in each lobe or in both lobes have now been produced. These monoferric hTFs will allow dissection of the contribution of each lobe to transferrin function. In addition, the construct completely lacking in the ability to bind iron in either lobe provides an opportunity to assess whether hTF has any other functions in addition to iron transport. Following insertion of the His-tagged hTF molecules into the pNUT vector, transfection into baby hamster kidney cells and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium and characterized with regard to their iron binding properties. Significant improvements over our previous protocol include: (1) addition of butyric acid at a level of 1mM which leads to a substantial increase in protein production (as much as a 65% increase compared to control cells); and (2) elimination of an anion exchange column prior to isolation on a Qiagen Ni-NTA column which makes purification of the His-tagged constructs faster and therefore more efficient. These improvements should be applicable to expression of other recombinant proteins in mammalian cells.
authors.affiliation: Department of Biochemistry, University of Vermont, College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405, USA. anne.mason@uvm.edu
authors.forename: Anne B
authors.initials: AB
authors.lastname: Mason
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Halbrooks
authors.forename: Julia R
authors.initials: JR
authors.lastname: Larouche
authors.forename: Sara K
authors.initials: SK
authors.lastname: Briggs
authors.forename: Marque L
authors.initials: ML
authors.lastname: Moffett
authors.forename: Jon E
authors.initials: JE
authors.lastname: Ramsey
authors.forename: Susan A
authors.initials: SA
authors.lastname: Connolly
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Smith
authors.forename: Ross T A
authors.initials: RT
authors.lastname: MacGillivray
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020148:Butyric Acid / Q000494:pharmacology; D002846:Chromatography, Affinity; D002999:Clone Cells; D006224:Cricetinae; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation; D006633:Histamine Antagonists / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D006633:Histamine Antagonists; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D020148:Butyric Acid; D007501:Iron