Research

Research at a glance

• Molecular interactions can occur between molecules belonging to different biochemical families (proteins, nucleic acids, lipids, carbohydrates, etc.) and also within a given family. Whenever such molecules are connected by physical interactions, they form molecular interaction networks (the "interactome") that are generally classified by the nature of the compounds involved. Despite impressive advances in high-throughput interactome mapping and disease gene identification, both the interactome and our knowledge of disease-associated genes remain incomplete.
• We have a strong interest in profiling the interactome of specific diseases, mainly characterized by the production of antibodies targeting self-antigens (autoimmune diseases and cancer). The final goal is to discover novel biomarkers that may prove useful for the diagnosis and the prognosis of the disease, and to guide therapeutic strategies.
• More in general, we are interested in developing innovative tools for the rapid interactome profiling

The research pipeline

• Phage display is a laboratory technique for the study of biomolecular interactions (protein-protein, protein-DNA, etc) that uses bacteriophages (viruses that infect bacteria) to connect proteins with the genetic information that encodes them.
• In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. These displaying phages can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those other molecules.
• In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection. Phage display has been widely used to select antibodies or short peptides from libraries, however, phage display with cDNA libraries has been rarely used due to the large number of non-functional clones present.
• This problem has been recently addressed by the concept of filtering DNA for the presence of open reading frames (ORFs), and a number of different approaches have been taken.
• In our lab, the cloning procedure is based on pPAO2, a display system developed by us, that directly filters DNA for ORFs within a phage display context, in such a way that they are amenable to subsequent selection or screening. In this vector, DNA fragments are cloned upstream of the β-lactamase gene and clones are selected for ampicillin resistance. After this step, referred to as ORF filtering, the β-lactamase gene is removed by Cre-lox mediated recombination and an in-frame ORF-g3p fusion product is displayed on the phage surface. This vector has a strong bias for ORFs corresponding to real genes rather than ORFs of no biological significance, indicating that the lactamase gene functions as a folding reporter.
• In our Discovery platform we apply a deep DNA sequencing strategy (NGS), first to the characterization of the cDNA phage display library, and subsequently to the identification of the proteins interacting with the bait of interest. The purpose is to follow the progress of the selection and analyze the whole output in a comprehensive manner, rather than performing random clone picking and enzyme-linked immunosorbant assay (ELISA).