Identifizierung und Charakterisierung von Metallchelat-bindenden Peptiden mittels Phage-Display

Recombinant proteins can be engineered for affinity to metal ions. This principle is exploited for the purification by immobilised affinity chromatography (IMAC). Fusion of a short sequence of 6 histidine residues to a protein is sufficient for binding to transition metal ions like Cu2+, Ni2+, Co2+ and Zn2+. A phage-displayed random 15-mer library was used to select peptides binding to transition metal ions as well as to hard lewis acids such as Fe3+ and Al3+. Different formats for the immobilisation of metal ions and stringency of selection were employed. Four rounds of panning were sufficient to enrich a phage population with specific binding to the given metal chelates. Peptides binding to transition metals with highest affinity contained 4 consecutive histidine residues, flanked with hydrophobic amino acids and proline. Specificity for the chelating support material correlated with relative position of the binding motif in the sequence and hydrophobicity. As an example, a helper phage carrying a transition metal binding peptide was constructed and shown to purify by IMAC. Panning on Fe3+ yielded positively charged peptides. The best binder contained 4 lysine and two histidine residues and was shown to bind to Ni2+ in absence of imidazole. Binding to Fe3+- IDA complexes could be efficiently competed with either primary amines, phosphate or pH 8 and higher. This bispecific peptide may serve for a cascade purification on Ni2+ and Fe3+ columns, yielding higher homogeneity. Furthermore, affinity purification with iron abrogates the tedious removal of contaminating toxic heavy metal ions in traditional IMAC.