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- Noppe, W, et al.
(författare)
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A probe for capture and Fe3+-induced conformational change of lactoferrin selected from phage displayed peptide libraries
- 2004
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Ingår i: Journal of Dairy Science. - 1525-3198. ; 87:10, s. 3247-3255
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Tidskriftsartikel (refereegranskat)abstract
- 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 (Kdsimilar to29 muM) and was also capable of inhibiting the binding of the phage to lactoferrin (IC(50)similar to17 muM), 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.
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- Noppe, W, et al.
(författare)
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Immobilised peptide displaying phages as affinity ligands purification of lactoferrin from defatted milk
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1101:1-2, s. 79-85
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Tidskriftsartikel (refereegranskat)abstract
- An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 mu m) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 mu m) phage particles as ligands without any risk of blocking the monolithic column. Bound lactoferrin was eluted using 1 M NaC1 with a purity of > 95%. The technique presents a good alternative to conventional immunoaffinity chromatography for purification of a protein of interest from complex samples due to (i) the robustness of the system in terms of recovery and ligand leakage and (ii) economical aspect in terms of low ligand cost.
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- Noppe, W., et al.
(författare)
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Macroporous monolithic gels, cryogels, with immobilized phages from phage-display library as a new platform for fast development of affinity adsorbent capable of target capture from crude feeds
- 2007
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 131:3, s. 293-299
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Tidskriftsartikel (refereegranskat)abstract
- Selected phage clones expressing a peptide with high binding affinity for recombinant human lactoferrin or von Willebrand factor (vWF) were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10–100 μm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 μm) phage particles as ligands without any risk of blocking the monolithic column. The macroporous monolithic columns were successfully used for the direct affinity capture of target proteins from particulate containing feeds like milk containing casein micelles and fat globules (1–10 μm in size) or even whole blood containing blood cells (up to 20 μm in size). The newly developed platform based on selected bacteriophages immobilized within macropores of the monolithic cryogels presents a convenient alternative to antibodies for fast and selective development of the specific adsorbent.
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