Creation of molecular switches by combinatorial protein engineering

Natural protein molecular switches are essential parts of the complex circuits that regulate cellular processes. For example, allosteric proteins are molecular switches in which modular functions are coupled. Artificial molecular switches, where chosen functions are coupled, might potentially have a wide area of applications, but a general engineering strategy to create such molecules does not exist and the rational design of such molecules by mimicking natural mechanisms is very challenging. In this work, combinatorial protein engineering was used to create molecular switches in which two chosen functions, maltose binding and beta-lactam hydrolysis, were coupled. These functions exist in two independent proteins in E. coli: maltose-binding protein (MBP) is responsible for the transport of maltose into the cytoplasm and beta-lactamase (BLA) is the enzyme that confers bacteria with resistance to beta-lactam antibiotics. These two functions were coupled by the covalent linkage of the two proteins. Diverse covalent fusions between the two domains were explored by in vitro recombination of their genes. This in vitro recombination procedure involved random circular permutation of bla gene and random domain insertion of bla into the gene that codes for MBP. From libraries of MBP-BLA hybrid proteins, several allosteric enzymes in which maltose binding modulated BLA activity were identified. In the best switch, maltose binding increased BLA activity by as much as 600-fold. This switch conferred E. coli cells with a maltose dependent resistance to beta-lactam antibiotics: the minimum inhibitory concentration of ampicillin was 16-fold higher in the presence of maltose than in the absence of maltose. Taking advantage of the fact that ligand binding was coupled to cell growth through enhanced BLA activity, MBP binding site was redesigned to bind sucrose as well as maltose.