title: Engineering of solvent-exposed loops in Escherichia coli beta-galactosidase.
abstract: The Escherichia coli beta-galactosidase is a high molecular mass tetrameric enzyme extensively used as a molecular marker. Despite its proven utility as a partner in fusion proteins, previous attempts to generate insertional mutants rendered inactive or poorly active enzymes, hampering its further engineering for the construction of multifunctional enzymes. We have explored several solvent-exposed loops on the tetramer, namely those spanning residues 246-254, 271-287, 578-584, 770-773, and 793-806, as acceptor sites to accommodate functional protein segments on the surface of active beta-galactosidase enzymes. An RGD-containing antigenic peptide positioned in these sites interacts efficiently with specific monoclonal antibodies as well as target integrins on the surface of mammalian cells. The resulting chimeric enzymes are soluble, stable, produced in high yields and enzymatically active. Moreover, the identified insertion sites could be appropriated for the design of promising beta-galactosidase-based molecular sensors.
authors.affiliation: Institut de Biologia Fonamental and Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
authors.forename: J X
authors.initials: JX
authors.lastname: Feliu
authors.forename: A
authors.initials: A
authors.lastname: Villaverde
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012997:Solvents; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D012997:Solvents; D001616:beta-Galactosidase