Chemical synthesis of a designed beta-protein through the flow-polyamide method.
A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a beta-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% beta 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 Ion Spray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.
Department of Biochemistry, P. Angeletti Institute for Research into Molecular Biology (IRBM), Pomezia, Rome, Italy.
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Bianchi
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Sollazzo
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Tramontano
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Pessi
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000302:isolation & purification; D002625:Chemistry, Organic / Q000379:methods; D007136:Immunoglobulins; D008667:Metalloproteins / Q000737:chemistry; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D009757:Nylons / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007136:Immunoglobulins; D008667:Metalloproteins; D008670:Metals; C081296:Minibody (WT); D009757:Nylons