ENGINEERING OF SOLVENT-EXPOSED LOOPS IN ESCHERICHIA-COLI BETA-GALACTOSIDASE

The Escherichia coli beta-galactosidase is a high molecular mass tetra meric enzyme extensively used as a molecular marker, Despite its prove n utility as a partner in fusion proteins, previous attempts to genera te insertional mutants rendered inactive or poorly active enzymes, ham pering its further engineering for the construction of multifunctional enzymes. We have explored several solvent-exposed loops on the tetram er, namely those spanning residues 246-254, 271-287, 578-584, 770-773, and 793-806, as acceptor sites to accommodate functional protein segm ents on the surface of active beta-galactosidase enzymes. An RGD-conta ining antigenic peptide positioned in these sites interacts efficientl y with specific monoclonal antibodies as well as target integrins on t he surface of mammalian cells. The resulting chimeric enzymes are solu ble, stable, produced in high yields and enzymatically active. Moreove r, the identified insertion sites could be appropriated for the design of promising beta-galactosidase-based molecular sensors. (C) 1998 Fed eration of European Biochemical Societies.