Directed evolution of protein switches and their application to the creation of ligand-binding proteins.
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.
Guntas
Gurkan
G
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Mansell
Thomas J
TJ
Kim
Jin Ryoun
JR
Ostermeier
Marc
M
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
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D013395:Sucrose; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1