Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition. Studies with recombinant ovotransferrins.
Different recombinant N-lobes of chicken ovotransferrin (oTF/2N) have been isolated from the tissue-culture medium of baby hamster kidney cells transfected with the plasmid pNUT containing the relevant DNA coding sequence. Levels of up to 40, 55 and 30 mg/1 oTF/2N were obtained for constructs defining residues 1-319, 1-332 and 1-337-(Ala)3 respectively. In addition, a full-length non-glycosylated oTF was expressed at a maximum of 80 mg/1 and a foreshortened oTF consisting of residues 1-682 was expressed at a level of 95 mg/l. These preparations were then used to produce, proteolytically, two different C-lobes (oTF/2C) comprising residues 342-686 and 342-682. The purified recombinant N-lobes (oTF/2N) are similar to the proteolytically derived half-molecule with regard to immunoreactivity and spectral properties; they show some interesting differences in thermal stability. A sequence analysis of the cDNA revealed six changes at the nucleotide level that led to six differences in the amino acid sequence compared with that reported by Jeltsch and Chambon [(1982) Eur. J. Biochem. 122, 291-295]. Electrospray mass spectrometry gives results consistent with these six changes. Interaction between the various N- and C-lobes was measured by titration calorimetry. Studies show that only those lobes that associate in solution are able to bind to the receptors on chick embryo red blood cells. These findings do not support a previous report by Oratore et al.
Department of Biochemistry, University of Vermont, Burlington 05405, USA.
A B
AB
Mason
R C
RC
Woodworth
R W
RW
Oliver
B N
BN
Green
L N
LN
Lin
J F
JF
Brandts
K J
KJ
Savage
B M
BM
Tam
R T
RT
MacGillivray
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002645:Chickens; D003207:Conalbumin / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D011990:Receptors, Transferrin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D003207:Conalbumin