| Abstract: | A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a β-sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70%β structure, Accordingly, it was anticipated, and actually found, to be a “difficult sequence” for all its length. Comparison of the standard, “minimum redundancy” protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full-length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity-type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi-dimensional purification scheme produced a highly homogeneous product, with the expected IonSpray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product. |
