A single mutation in the inducer-binding site of the lac repressor induces tighter control of protein expression levels.View all posters
University of Cambridge, United Kingdom
The lac repressor (LacI) is one of the most widely used systems for the overexpression of heterologous proteins in Escherichia coli. The protein works as a homodimer that recognizes and binds to a specific sequence on the DNA (operator DNA). Two copies of the operator DNA favour the formation of a dimer of dimers that causes the DNA to loop, inhibits binding of the RNA polymerase and results in protein production being switched off. In nature, binding of allolactose induces rearrangements in the protein structures that result in DNA release and de-repression of transcription. Instead, the non-hydrolysable mimic isopropyl -D-1-thiogalactopyranoside (IPTG) is used for protein over-expression as it efficiently triggers DNA release but remains at high concentration inside the cell. With the aim of altering its responsiveness to IPTG, we mutagenized the inducer-binding pocket of LacI. One mutant showed efficient protein production in the presence of IPTG, but at the same time significantly reduced basal expression levels in the absence of the inducer. GFP expression was used to characterise this mutant and expression kinetics revealed an up to 100-fold decrease in basal expression levels at both 37 °C and 25 °C. As GFP production levels still reach those of wild type LacI, the mutant effectively has a wider dynamic range. Thermal shift assays and isothermal calorimetry showed that, compared to wt, the mutant has lower affinity for IPTG and higher for the operator. When used for the expression of a toxic protein, faster E. coli growth rates are observed with mutant LacI as compared to wt. A single mutation in the LacI inducer-binding site generated a variant with a wider dynamic range and reduced leakiness that is suitable for heterologous protein production. The characterisation of its biological and biophysical behaviour reveals key factors for the function of the lac repressor.