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.
Bianchi
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E
Department of Biochemistry, P. Angeletti Institute for Research into Molecular Biology (IRBM), Pomezia, Rome, Italy.
Sollazzo
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M
Tramontano
A
A
Pessi
A
A
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