A probe for capture and Fe3+-induced conformational change of lactoferrin selected from phage displayed peptide libraries.
Linear pentadecamer and cyclic hexamer peptide phage libraries were used to isolate phage clones with binding affinity toward lactoferrins purified from human and bovine milk. Phage clones with high specificity toward lactoferrin were selected with different binding strengths depending on the sequence of the peptide displayed by the phage. Phages coated to a microtiterplate were able to capture lactoferrin from crude milk samples without prior treatment. One of the selected sequences, EGKQRR, failed to bind to lactoferrin. In contrast, a branched tree-peptide bearing 4 EGKQRR sequences did bind to lactoferrin (Kd approximately 29 microM) and was also capable of inhibiting the binding of the phage to lactoferrin (IC(50) approximately 17 microM), indicating that avidity was important. Unexpectedly, the affinity of the phage for lactoferrin was influenced by the amount of bound Fe(3+), with a much lower affinity when lactoferrin was saturated with Fe(3+) as compared with the iron-depleted or partially saturated (natural) lactoferrin. As the phage does not bind to the Fe(3+)-binding site, the difference in binding affinity is due to differences in conformation of lactoferrin induced by Fe(3+). These results demonstrate that avidity or multipoint attachment and Fe(3+)-induced conformational changes play an important role in the binding of the selected phage to lactoferrin. Thus, we could demonstrate that, by the use of selected phage clones, we are able not only to detect lactoferrin, but also to capture lactoferrin from crude milk samples. Furthermore, the extent of phage binding provides additional information about the iron content and the concomitant conformation of lactoferrin.
Noppe
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Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, E. Sabbelaan 53, B-8500 Kortrijk, Belgium.
Vanhoorelbeke
K
K
Galaev
I Y
IY
Mattiasson
B
B
Deckmyn
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000378:metabolism; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D005290:Ferric Compounds / Q000494:pharmacology*; D007781:Lactoferrin / Q000737:chemistry* / Q000378:metabolism; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation / Q000187:drug effects*; D020349:Surface Plasmon Resonance
D005290:Ferric Compounds; D019151:Peptide Library; D007781:Lactoferrin