Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins

Protein Expression and Purification
Anne B MasonRoss T A MacGillivray

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% i...Continue Reading

References

May 1, 1975·Nature·J V Princiotto, E J Zapolski
Nov 26, 1976·Biochimica Et Biophysica Acta·D G Makey, U S Seal
Apr 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·A Dautry-VarsatH F Lodish
Apr 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·R D KlausnerK R Bridges
Apr 30, 1981·Biochemical and Biophysical Research Communications·D A Baldwin, D M de Sousa
Feb 1, 1980·The Biochemical Journal·J Williams, K Moreton
Nov 24, 1995·Journal of Molecular Biology·H KurokawaM Hirose
Jan 18, 1994·Proceedings of the National Academy of Sciences of the United States of America·J P BasilionW H Burgess
Aug 15, 1997·The Biochemical Journal·A B MasonR T MacGillivray
Jul 27, 2001·The International Journal of Biochemistry & Cell Biology·P AisenM Wessling-Resnick
Dec 26, 2001·Acta Crystallographica. Section D, Biological Crystallography·D R HallP F Lindley
Dec 18, 2002·Proceedings of the National Academy of Sciences of the United States of America·Mahantesh S NavatiJoel M Friedman
Mar 6, 2003·Vox Sanguinis·Jaakko ParkkinenLeila Sahlstedt
Mar 26, 2003·Protein Expression and Purification·Olga Zak, Philip Aisen
Sep 10, 2003·Biochemical and Biophysical Research Communications·Hitoshi OgawaDavid G Binion
Oct 22, 2003·Molecular Immunology·Sebastien Farnaud, Robert W Evans
Dec 24, 2003·PLoS Biology·Anthony M GiannettiPamela J Björkman
Feb 26, 2004·Cell·Yifan ChengThomas Walz

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Citations

Apr 23, 2013·Analytical and Bioanalytical Chemistry·Grzegorz MajkaMałgorzata Brindell
Mar 18, 2009·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Shaina L Byrne, Anne B Mason
Feb 14, 2012·Nature·Nicholas NoinajSusan K Buchanan
Apr 21, 2010·Proceedings of the National Academy of Sciences of the United States of America·Rachael LeverenceIgor A Kaltashov
Jul 27, 2011·Proceedings of the National Academy of Sciences of the United States of America·Brian E EckenrothAnne B Mason
Jul 10, 2010·The Journal of Biological Chemistry·Suzan H M RooijakkersVictor Nizet
Feb 9, 2010·Bioscience, Biotechnology, and Biochemistry·Kimihiko MizutaniBunzo Mikami
Mar 1, 2013·Metallomics : Integrated Biometal Science·Gauthier J-P DeblondeRebecca J Abergel
May 8, 2010·Protein Expression and Purification·Deshui ZhangNing Huang
Jun 24, 2010·Protein Science : a Publication of the Protein Society·Brian E EckenrothStephen J Everse
Nov 17, 2009·Protein Science : a Publication of the Protein Society·Nicholas G JamesDavid M Jameson
May 13, 2008·Analytical Biochemistry·Nicholas G James, Anne B Mason
Aug 23, 2011·Biochimica Et Biophysica Acta·Alex D SheftelPrem Ponka
Jun 28, 2011·Biochimica Et Biophysica Acta·Ashley N SteereAnne B Mason
Dec 30, 2014·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Guinevere MathiesEdgar J J Groenen
Jan 17, 2013·Journal of the American Chemical Society·Christine S ScheveJeanne C Stachowiak
Mar 1, 2012·The Journal of Physical Chemistry. B·Rajesh Kumar, A Grant Mauk
Nov 5, 2016·Biochimica Et Biophysica Acta·Amel HamdiPrem Ponka

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