Enginyeria d'interaccions moleculars intracèl·lulars; aplicació al disseny de biosensors i al control de la desagregació proteïca

The global objective of this proposal is the mechanistic and functional analysis of two kind of intracellular intermolecular interactions:those involving recombinant ligand that modulate enzymatic activities as a result of subtle conformational changes, and those occuring between misfolded polypeptide chains that promote their deposition as aggregates or inclusion bodies. In this context, we propose four partial, clearly defined objectives (two for each area): 1. To improve the function and robustness of enzymatic biosensors based in \i E. coli\i0 \beta-galactosidase (as displaying foreign peptide antiogens) and the identificaction of new insertion sites (or combinations of different sites) for peptide accommodation. To increase the ratio signal/background and biosensor sensitivity by optimizing the assay conditions and through the engineering of the enzyme. We expect to gain both scientific and methodological basis for a better rational design of biosensor devices for diagnosis, antibody detection and targeting of specific ligands, paying attention to the possible automatic performance of the homogeneous assay. 2. To evaluate the performance of such enzymatic sensors intracellularly, to design biological systems allowing the identification and positive selection of specif ligands among libraries of antibodies (or other ligands). The objective is to obtein an intracellular detection system based on \beta-galactosidase, responsive to specific antibodies with an increase in the enzymatic activity. In an appropriate genetic background (LacZ-) and in presence of lactose as carbon source, it should allow a differential growth of positive bacterial clones. 3. To perform an exhaustive proteomic analysis of inclusion body formation..4. To determinate the particular cell mechanisms involved in protein solubilizationfrom inclusion bodies