The present proposal aims at the characteriazion of the 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 by means of state-of-the-art spectroscopic and computational approaches.
Two main aspects of originality characterize the proposed research programme.
The first point of originality concerns the complete integration of the partners with different expertise that assure a multi-scale approach aimed to provide a detailed understanding of the mechanisms involved in the intein splicing Process.
That is, M.D. and E.B. expertise on computational modeling will be merged with spectroscopic characterization of these systems, in order to provide a complete and accurate description of the intein mediated protein splicing reaction.
The second point is the potential use of this kind of systems in the market of protein purification and isolation, possibly bringing important benefits to society such as the possibility to identify, develop and produce new bio-products, especially in the area of human health.
Many bio-therapeutic candidates that are not further developed due to high purification cost, could be easily produced by such an approach. In addition, inteins-based purification systems are environmentally friendly method for protein purification since it does not use harmful reagents, it does not generate toxic residues such as heavy metals used in other purification methods, and reduces the amount of resources needed (water, energy, etc), thus representing a valid pulse on a "green" approach in chemistry.
Possible progress beyond the State of the Art
The possibility to characterize the mutual orientation of the N- and C- intein domains when they bind each other and how the structure and dynamics of the intein complex is modified by the presence of the protein of interest (POI) would highly improve our understanding on the cleavage process and on current limitations in the intein purification systems. For example, as recently observed [Guan2013], the possible steric hindrance of the critical interaction between the N- and C- domains might result in a remarkable loss of purification efficiency.
Furthermore, an additional trigger mechanism able to initiate the trans-splicing process (Berrade2010) could be studied by UV-spectroscopy in model systems and these data combined with the theoretical-computational approach.
References
Guan D, Ramirez M, Chen, Z. Biotechnology and Bioengineering. 2013; 110(9): 2471-2481.
Berrade L, Kwon Y, Camarero JA. Photomodulation of protein trans-splicing through backbone photocaging of the DnaE split intein. Chembiochem. 2010 Jul;11(10) 1368-1372. doi:10.1002/cbic.201000157.