The making of the minibody: an engineered beta-protein for the display of conformationally constrained peptides.
Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost the drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue beta-protein aimed at retaining some desirable features of immunoglobulin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of the main chain conformation of the same regions, based on the 'canonical structures' model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical synthesis and expression in E. coli and characterized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low solubility. We were able to overcome this problem both by further mutagenesis of the framework and by addition of a solubilizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.
Tramontano
A
A
Department of Biocomputing, Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia, Roma, Italy.
Bianchi
E
E
Venturini
S
S
Martin
F
F
Pessi
A
A
Sollazzo
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003090:Coliphages; D004926:Escherichia coli; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D007163:Immunosorbent Techniques; D015850:Interleukin-6 / Q000276:immunology*; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
D007135:Immunoglobulin Variable Region; D015850:Interleukin-6; D008670:Metals; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins