Abstract
Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These ‘phenotypic switch’ genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.
Highlights
IntroductionThe regulation of cellular protein activity occurs through many mechanisms including the regulation of production (i.e. transcription/translation), the targeting to specific cellular compartments, the interaction with other molecules (i.e. inhibitors, activators, and allosteric effectors) and the regulation of degradation
The regulation of cellular protein activity occurs through many mechanisms including the regulation of production, the targeting to specific cellular compartments, the interaction with other molecules and the regulation of degradation
Using a two-tiered genetic selection designed to identify allosteric b-lactamases that are activated by maltose, we isolated 34 library members from a library in which random circular permutations of TEM1 b-lactamase gene were inserted in place of codon 317 of the gene encoding E. coli maltose binding protein (MBP) [5]
Summary
The regulation of cellular protein activity occurs through many mechanisms including the regulation of production (i.e. transcription/translation), the targeting to specific cellular compartments, the interaction with other molecules (i.e. inhibitors, activators, and allosteric effectors) and the regulation of degradation. We have pursued a directed evolution approach to this design problem, creating libraries of random insertions of one domain into the other and using random circular permutation of the inserted domain to vary the fusion location [2,3,4]. Such an approach requires a well-designed selection/screening method in order to find those rare protein switches among the majority of constructs that lack effector-dependent activity. Since regulation of cellular protein activity can occur by many mechanisms other than effectorinduced modulation of a protein’s specific activity, such selections might result in the identification of gene fusions that confer switching behavior to cells by means other than allostery
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