A molecular switch created by in vitro recombination of nonhomologous genes.
We have created a molecular switch by the in vitro recombination of nonhomologous genes and subjecting the recombined genes to evolutionary pressure. The gene encoding TEM1 beta-lactamase was circularly permuted in a random fashion and subsequently randomly inserted into the gene encoding Escherichia coli maltose binding protein. From this library, a switch (RG13) was identified in which its beta-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose. Upon removal of maltose, RG13's catalytic activity returned to its premaltose level, illustrating that the switching is reversible. The modularity of RG13 was demonstrated by increasing maltose affinity while preserving switching activity. RG13 gave rise to a novel cellular phenotype, illustrating the potential of molecular switches to rewire the cellular circuitry.
Guntas
Gurkan
G
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Mitchell
Sarah F
SF
Ostermeier
Marc
M
D000818:Animals; D029968:Escherichia coli Proteins / Q000235:genetics*; D005812:Genes, Switch; D008958:Models, Molecular; D033902:Periplasmic Binding Proteins / Q000235:genetics*; D015202:Protein Engineering; D011995:Recombination, Genetic; D017385:Sequence Homology; D001618:beta-Lactamases / Q000235:genetics*
D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D001618:beta-Lactamases