Photochemical Activation of Protein Expression in Bacterial Cells

Small-molecule-inducible gene-expression systems are available in a variety of cell types including bacterial cells, yeast cells, and mammalian cells. They have been used for conditional protein expression in higher organisms ranging from plants to mice. These systems are typically composed of a natural receptor taken from one organism and transferred into a second organism, in conjunction with a natural or unnatural small-molecule ligand that is orthogonal to all endogenous molecules of the organism of interest. Exposure of the organism to the small molecule typically activates gene expression, a process that has found application in the production of recombinant proteins, the programing of biological processes, and the study of gene function. Although this technique enables temporal control over gene function on a minute-to-hour timescale, it does not permit spatial control. A highly efficient way to simultaneously achieve spatial and temporal control over biological processes is the application of photocaging. The term “caging” defines the installation of a photoremovable group on a biologically active molecule, thus rendering the molecule inactive. Caging groups have been installed on small molecules, oligonucleotides, peptides, and proteins. Irradiation with UV light removes the caging group and restores biological activity. Recently, this approach has been applied to the light activation of small-molecule inducers of protein expression. Both reported systems, that is, the doxycycline and the nuclear hormone (for example, estradiol) conditional-geneexpression systems, are restricted to eukaryotic cells. Here we report a light-inducible gene-expression system which can be used in bacterial cells, plants, and mammalian organisms. It is based on the lactose (lac) repressor which binds to the lac operator (lacO), thereby inhibiting RNA polymerase from performing gene transcription. In presence of the small-molecule effector isopropyl-b-d-thio-galactoside (IPTG), the repressor is released from the DNA through an allosteric binding event, which results in a conformational change and leads to gene expression (Figure 1). The crystal structure of the LacI/IPTG complex (Protein Data Bank file no. 1LBH) reveals interactions between the small molecule and the protein in a tight binding pocket, as well as four essential hydrogen bonds between the 4-OH group and Arg197, the 3-OH group and Arg197, and also the 2-OH group and Asp274 and Asn246 (see the Supporting Information). We speculated that disruption of the hydrogen-bond network through the installation of a sterically demanding caging group on IPTG would inhibit the formation of the LacI/IPTG complex and thereby inhibit gene expression. If the installed group is removable through irradiation with UV light, IPTG can be generated in a spatiotemporal manner, thereby enabling spatiotemporal control over protein expression. In order to achieve this, the caged IPTG 1 was synthesized in a single step (78% yield) by the reaction of IPTG with 6-nitropiperonal (see the Experimental Section), which furnished selective dioxolan formation at the 4and 6-hydroxy groups. This is in accordance with the results of NMR experiments and previous observations in similar reactions. The caged IPTG 1 is nontoxic to bacterial cells and no degradation was observed under physiological conditions. Irradiation of an aqueous solution of 1 ( 0.5 mm, e365= 4533 cm m ) with nonphotodamaging UV light (handheld UV lamp, 365 nm, 0.5 Wcm ) for 5 min leads to quantitative formation of the ester 2, as a 1:1 mixture of regioisomers (4-OH/6-OH, Scheme 1 shows the 4-OH ester). The half life for the conversion 1!2 after irradiation depends on the concentration of 1 and amounts to 11 s at a concentration of 0.1 mm, 5.1 min at 0.5 mm, or 11.8 min at 1.0 mm (see the Supporting Information). This facile photolytic conversion of a 1,3-dioxane is in remarkable contrast to previous observations with bromohydroxycoumarin caged diols. The quantum yield (f= 0.131) for the photochemical conversion of 1 into 2 has been determined by 3,4-dimethoxynitrobenzene actinometry. The ester 2 is stable in an aqueous solution; however, as observed for other carbohydrate esters, 2 is hydrolyzed to IPTG by the esterases found in a cellular environment (t1/2= 63 min 2 min, see the Supporting Information). The growth of bacterial cells exposed to 0.5 mm concentrations of 1 and 2 revealed that the compounds do not reduce growth rates, are nontoxic, and are easily taken up by the cells (see the Supporting Information). Figure 1. IPTG-induced expression of an open reading frame (ORF) through formation of the IPTG/lac repressor complex.