title: Directed evolution of protein switches and their application to the creation of ligand-binding proteins.
abstract: We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 beta-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of beta-lactam hydrolysis. Some of these MBP-BLA switches were effectively "on-off" in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth/no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 x 10(6) variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a "sucrose-binding protein," illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Gurkan
authors.initials: G
authors.lastname: Guntas
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Mansell
authors.forename: Jin Ryoun
authors.initials: JR
authors.lastname: Kim
authors.forename: Marc
authors.initials: M
authors.lastname: Ostermeier
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics* / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D019020:Directed Molecular Evolution; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005812:Genes, Switch / Q000235:genetics; D008024:Ligands; D058619:Maltose-Binding Proteins; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D013395:Sucrose / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D013395:Sucrose; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1