A new method for obtaining human transferrin C-lobe in the native conformation: preparation and properties.
Eukaryotic transferrins comprise a class of bilobal iron-binding proteins in which each lobe carries a single binding site. Although expression of full-length transferrins and their N-terminal lobes, in wild-type and mutated forms, has been successfully accomplished by several laboratories, expression of C-lobes has been much less satisfactory. A possible explanation of the difficulty is that proper folding of the C-lobe, with its 11 disulfide bonds, depends on prior synthesis and proper folding of the N-lobe. We have therefore developed a new strategy, introducing a specific factor Xa cleavage site in the interlobe-connecting strand to permit separation of the lobes after expression of the full-length protein. The resulting protein was expressed in satisfactory yield, >20 mg/L, and could be easily and completely cleaved to yield two distinguishable fragments representing N- and C-lobes, respectively. Retaining the glycosylation sites, found only in the C-lobe, made it possible to separate the fragments from each other by ConA affinity chromatography. The isolated C-lobe so obtained displayed spectroscopic and kinetic features of the C-lobe in native transferrin and was competent as an iron donor for K562 cells to which it bound in saturable fashion inhibitable by native diferric transferrin. Since the N-lobe by itself will neither bind nor donate iron to cells, the primary receptor-recognition site of transferrin resides in its C-lobe.
Zak
Olga
O
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Aisen
Philip
P
D000818:Animals; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D004578:Electron Spin Resonance Spectroscopy; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism; D020014:K562 Cells / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron; D015951:Factor Xa