A poly-His tag method for obtaining the C-terminal lobe of human transferrin.
Human serum transferrin is an essential bilobal protein that transports iron in the circulation for delivery to iron-requiring cells. Obtaining the C-terminal lobe of human transferrin in verified native conformation has been problematic, possibly because its 11 disulfide bonds lead to misfolding when the lobe is expressed without its accompanying N-lobe. A recently reported method for preparing the C-lobe free of extraneous residues, with normal iron-binding properties and capable of delivering iron to cells, makes use of a Factor Xa cleavage site inserted into the interlobal connecting strand of the full-length protein. An inefficient step in this method requires the use of ConA chromatography to separate the cleaved lobes from each other, since only the C-lobe is glycosylated. Inserting a 6-His sequence near the start of the N-lobe enhances recovery of the recombinant transferrin from other proteins in the culture medium of the BHK21 cells expressing the mutant transferrin. The new procedure is more economical in time and effort than its predecessor, and offers the additional advantage of isolating C-lobe expressed with or without its glycan chains.
Zak
Olga
O
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Aisen
Philip
P
D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D033863:Transferrin-Binding Proteins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014168:Transferrin; D033863:Transferrin-Binding Proteins; D006639:Histidine