Maurizio Sollazzo

The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6.

A major challenge in basic and applied biological research is the engineering of small proteins with pre‐determined structures and novel functions. In a limited number of cases, this has been achieved by de novo design. An alternative combinatorial approach is based on the construction of large libraries of random peptides and on methods for the selection of the desired molecules. Here we describe a successful combination of both the rational design and the combinatorial approaches for developing proteins with useful biological functions, in this case the construction of a specific inhibitor of the cytokine human interleukin‐6. In previous work, the ‘minibody’, a 61 residue polypeptide consisting of a beta‐pleated framework and two hypervariable regions, was designed, synthesized and expressed on f1 phage surface. We report the construction of a repertoire of 50 million minibodies displayed on phage in which the hypervariable regions have been randomized. One polypeptide which binds tightly and specifically to human interleukin‐6 was isolated from this collection of minibody mutants. This particular minibody is an effective inhibitor of the cytokines biological activity. The approach described here could in principle be applied to other molecular targets.

Identification of biologically active peptides using random libraries displayed on phage

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Inhibition of the Hepatitis C Virus NS3/4A Protease THE CRYSTAL STRUCTURES OF TWO PROTEASE-INHIBITOR COMPLEXES

The hepatitis C virus NS3 protein contains a serine protease domain with a chymotrypsin-like fold, which is a target for development of therapeutics. We report the crystal structures of this domain complexed with NS4A cofactor and with two potent, reversible covalent inhibitors spanning the P1–P4 residues. Both inhibitors bind in an extended backbone conformation, forming an anti-parallel β-sheet with one enzyme β-strand. The P1 residue contributes most to the binding energy, whereas P2–P4 side chains are partially solvent exposed. The structures do not show notable rearrangements of the active site upon inhibitor binding. These results are significant for the development of antivirals.

Peptide and protein display on the surface of filamentous bacteriophage.

The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics. Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences. Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques. The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines.

A novel ADP- and zinc-binding fold from function-directed in vitro evolution

A great challenge to biologists is to create proteins with novel folds and tailored functions. As an alternative to de novo protein design, we investigated the structure of a randomly generated protein targeted to bind ATP. The crystal structure reveals a novel α/β fold bound to its ligand, representing both the first protein structure derived from in vitro evolution and the first nucleotide-binding protein stabilized by a zinc ion.

Characterization of human ferritin H chain synthetized in Escherichia coli

We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2. The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins. All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified. We have shown that the ferritin synthesized in E. coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.

Idiotype Vaccines by Antibody Engineering: Structural and Functional Considerations

The idea of utilizing antibodies as noninfectious vaccines originates from the concept of idiotypes as imperfect chemical copies of antigens (1, 2) and is based on the property that antibodies can be immunogenic (3). This has been demonstrated in heterologous (4), homologous (3), syngeneic (5), and autologous (6) systems. Historically, the existence of immunoglobulin molecules related to antigenic determinants, not by fitting them but rather by resembling them, was first hypothesized by Lindemann (1). Borrowing his words there exist two different varitypes* related to one particular antigenic determinant: a varitype leading to a combining site matching that determinant, and which is conventionally called antibody; another varitype leading to an idiotypic site cross-reacting with that determinant, and for which we propose the name of homobody. Accordingly, antigenic determinants are projected into the world of immunoglobulin molecules in two manners: as negative images, these define antibodies; as positive images, these define homobodies.