IPTG is a chemical inducer of gene expression used for the production of recombinant proteins from bacterial hosts and mammalian cells. The IPTG-inducible genetic systems are versatile and controllable tools for production of proteins for therapeutic, diagnostic or research purposes.
Mechanism of action in biological systems
IPTG is a molecular mimic of lactose metabolite allolactose. Lactose is an important carbon source for bacteria and has a regulatory role in the expression of lactose-metabolising proteins encoded by the lac operon. Given the molecular similarity between allolactose and IPTG, the latter is being used in molecular biology to activate lactose-responsive genetic elements.
Lac operon structure and regulation
The lac operon is coding for three structural genes involved in lactose catabolism. Gene lacZ is coding for β-galactosidase, which breaks down the lactose to galactose and glucose. LacY codes for a galactoside permease responsible for import of lactose into the cells. The lacA gene encodes a β-galactoside transacetylase involved in sugar metabolism. Upstream structural genes there are lac promoter (P) and operator (O) responsible for control of gene expression from the lac operon (figure 1).
In the absence of lactose, the lac repressor LacI is bound to the lac operator preventing the RNA polymerase binding to the promoter hence blocking gene transcription (figure 1A). Lactose addition to the cells inactivates the LacI repressor, causes its disassociation from the DNA strand and allows the RNA polymerase binding to the promoter (figure 1B). This initiates the transcription of lac genes and production of encoded proteins.
Figure 1. Schematic representation of the lac operon in its repressed (1A) and de-repressed state (1B).
Applications - in bacteria
Escherichia coli is the most common prokaryotic host for the overexpression of proteins. Herein, several elements of the lac operon have been rearranged to allow IPTG-inducible protein production. Commercially available recombinant strains of E. coli are carrying lac promoter-operator elements on its chromosome fused to a gene coding for T7 RNA polymerase (DE3 series strains). The repressor gene lacI is located on the E. coli chromosome and/or on the plasmid. The plasmid contains T7 RNA polymerase promoter followed by a cloning site for the insertion of genes of interest. Addition of IPTG to E. coli carrying a recombinant plasmid causes de-repression of lac operator allowing the expression of T7 polymerase from chromosome leading to the initiation of gene transcription from T7 promoter on the plasmid and consequently, protein expression (figure 2).
Figure 2. Schematic representation of the IPTG inducible genetic system for overexpression of proteins in E. coli.
Applications - in mammalian cells
IPTG-inducible systems have been adapted for the use in eukaryotic cells for the control of gene expression using similar approach as described above. The eukaryotic system consists of 3 vectors: an eukaryotic Lac-repressor-expressing vector and two eukaryotic lac-operator-containing vectors containing a gene of interest.