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A Probe for Capture and Fe³⁺-Induced Conformational Change of Lactoferrin Selected from Phage Displayed Peptide Libraries

¹ Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, E. Sabbelaan 53, B-8500 Kortrijk, Belgium
² Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-22100, Lund, Sweden

Corresponding author: H. Deckmyn; e-mail: hans.deckmyn{at}kulak.ac.be.

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~29 µM) and was also capable of inhibiting the binding of the phage to lactoferrin (IC₅₀~17 µM), indicating that avidity was important. Unexpectedly, the affinity of the phage for lactoferrin was influenced by the amount of bound Fe³⁺, with a much lower affinity when lactoferrin was saturated with Fe³⁺ as compared with the iron-depleted or partially saturated (natural) lactoferrin. As the phage does not bind to the Fe³⁺-binding site, the difference in binding affinity is due to differences in conformation of lactoferrin induced by Fe³⁺. These results demonstrate that avidity or multipoint attachment and Fe³⁺-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.

Key Words: lactoferrin • phage display • iron binding • avidity

Abbreviation key: apo-lactoferrin = iron-depleted lactoferrin, BovLF = bovine lactoferrin, HRP = horse-radish peroxidase, HuLF = human lactoferrin, TPBS = 0.1% Tween-20 in phosphate-buffered saline.

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