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.
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, A.S. expertise on design, expression and characterization of split inteins will be merged with computational modeling and spectroscopical characterization of these systems, in order to provide a complete and accurate description of the inteins function and behavior on a multi-scale level, ranging from the atomic detail to the protein purification reactions.
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.
References
Guan D, Ramirez M, Chen, Z. Biotechnology and Bioengineering. 2013; 110(9): 2471-2481.