Ricerc@Sapienza

The present proposal aims at generating experimental and computational studies to delineate intein-mediated protein splicing mechanism. Inteins are a widely distributed family of self-splicing proteins, just like RNA splicing, with an ability to excise themselves from flanking host protein regions, the exteins, in an autocatalytic manner with remarkable precision, ligating flanked host protein fragments. Split inteins are an especially interesting subfamily of inteins. In this case intein is split into two pieces and splicing only occurs upon reconstitution of these fragments: they generate a single protein chain from two individual polypeptides. Protein-splicing technology is already adapted to a wide range of applications, starting with untagged protein purification, site-specific protein labeling, protein biotinylation, isotope incorporation, peptide cyclization, as antimicrobial target, and so on.
However, structural features of inteins influencing the protein splicing reaction steps, controlling their efficiency and general applicability are poorly understood. We aim at generating a comprehensive analysis of the behaviour of protein splicing reaction/components in order to shedding light on the role played by the split-inteins. To achieve this aim, state-of-the-art biochemical and computational approaches will be combined to decipher mainly two poorly understood features of intein activity. The specific aims include: 1) bringing light to the role of the bioengineered proteins fused to inteins to delineate the best strategies for the design of optimal protein constructions and make protein splicing universal. 2) defining a protein design program by using advanced modelling to understand at atomic level protein splicing catalytic process and to render the reaction more efficient and general. Our results should contribute to forthcoming efforts in the development of intein-based biotechnology.