IPTG is the usual abbreviation for isopropyl b-D-thiogalactopyranoside. It is typically produced using D-galactose derived from milk lactose. Carbosynth also manufactures a grade that is of plant origin and therefore does not have the usual TSE/BSE issues. We call this grade “IPTG non-animal origin” and it is available as a free flowing crystalline powder in a range of packaging from 50 g to 20 kg drums.
IPTG is a molecular biology reagent, with multiple applications based on its ability to induce expression of genes under the control of the lac operon in some enteric bacteria, including E. coli.1
The lac operon is required for transport and metabolism of the disaccharide lactose in these bacteria, and is regulated by multiple factors including the availability of glucose and lactose. Bacterial cells can use lactose as a source of energy by producing the enzyme b-galactosidase which hydrolyses lactose into its constituent monosaccharides, galactose and glucose, Scheme 1.
Production of b-galactosidase when no lactose is available to the cell, or when glucose is available as an alternative energy source, would be unnecessary and inefficient and the lac operon uses a control mechanism to ensure that this enzyme and others necessary for lactose utilization are only produced when appropriate. An intracellular regulatory protein, lac repressor, inhibits production of b-galactosidase in the absence of lactose, by binding tightly to a short DNA sequence in the lac operon, known as the lac operator. Binding of the repressor protein in this fashion interferes with binding of the enzyme RNA polymerase to the DNA, resulting in the inhibition of production of proteins encoded by the operon. When cells are grown in the presence of lactose, a metabolite called allolactose (an isomeric form of lactose where the constituent monosaccharides are linked through the 1 and 6 positions, rather than 1 and 4 as in lactose – Scheme 2) binds to the repressor and causes a change in its shape. This change renders the repressor unable to bind to the operator, which allows the RNA polymerase enzyme to transcribe the lac genes and produce the encoded proteins.
IPTG finds utility in physiological work as an inducer of the lac operon. Acting as a mimic of allolactose, it binds to the repressor and inactivates it. Unlike allolactose, IPTG is not a substrate for b-galactosidase, as the carbon-sulfur bond cannot be cleaved by the enzyme, so its concentration remains constant. Intake of IPTG into a bacterial cell is dependent upon lactose permease, an enzyme which transports the disaccharide across the cell membrane into the cellular interior.
In cloning experiments one of the lac operon genes (lac Z), can be replaced with another gene and IPTG is then used to induce gene expression and production of the target protein. IPTG is typically an effective inducer in the concentration range 100 mM to 2 mM.
X-Gal (5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside; Carbosynth product code EB06680) is frequently used in molecular biology applications to indicate the activity of b-galactosidase. X-Gal is cleaved by the enzyme to yield galactose and 5-bromo-4-chloro-3-hydroxyindole, which then oxidises into an insoluble blue product. X-Gal and IPTG as an inducer of b-galactosidase are used in combination on an agar medium on a culture plate, and bacterial colonies with a functional lacZ gene can be readily identified by their blue colour.