Detection and Purification of lac Repressor
The previous section showed that wild-type lac repressor in crude extracts of cells was not likely to produce a detectable signal in the equilibrium dialysis assay. Therefore Gilbert and Müller-Hill isolated a mutant repressor that bound IPTG more tightly than the wild-type repressor. Crude extracts made from this strain showed an excess of counts in the dialysis sack. The excess was barely detectable; nonethe - less it was statistically significant, and fractionation of the extract yielded a protein sample with an easily detectable excess of counts.
Once the assay of lac repressor detected something, it was of great importance to prove that the origin of the signal was repressor and not something else. The proof used the tight-binding mutant. First, the tight-binding mutant was used to develop a partial purification of repressor so that a fraction could be obtained in which the signal was readily detectable. Then this same purification procedure was used to obtain a similar fraction from wild-type cells. This too generated a significant signal. The proof came with the demonstration that the apparent dissociation constants for IPTG in the fraction from the mutant and the wild-type were different. This was simply done by performing the dialysis on a series of samples at different concentra-tions of IPTG. The sample obtained from the mutant bound IPTG more tightly. That is, it had a smaller KD, than the wild-type (Fig. 11.5). As the only difference between the mutant and the wild-type was a mutation in the lacI gene, the signal in the assay was from lac repressor.
The definitive detection of repressor opened the door to biochemical studies. First, with an assay, the repressor could in principle be purified and used in biochemical studies probing its mechanism of action. Second, it was possible to attempt to isolate mutants that synthesized elevated quantities of repressor so as to ease the burden of purification. With an assay, such candidates could be identified.
Figure 11.5 Results of equilib-rium dialysis at different IPTG concentrations of wild-type re-pressor and a tight-binding mutant repressor. Rearranging the binding equation derived in the text yields a form convenient for plotting data, RI = R - [K ×(RI)/I]. In this form thevalue of RI when (RI)/I = 0 yields the concentration of R molecules capable of binding I and the slope of the binding curve gives K.
A mutation rendering lac repressor temperature-sensitive was used in the isolation of mutants possessing higher levels of repressor. Cells were grown at a temperature just high enough to inactivate most of the temperature-sensitive repressor. Consequently the lac operon was no longer repressed, and the cells expressing the lac operon were then killed. The survivors, which were able to repress the operon, could be of two types. Either the repressor was altered so that it could repress at
the elevated temperature or more repressor was being synthesized. The two types of mutants could easily be distinguished with the equilibrium dialysis assay, and an overproducing mutant was identified.
The selection for the loss of constitutivity in the scheme described above used yet another lactose analog, TONPG (o-nitrophenyl-1-thio-β-D-galactoside). This inhibits growth when it is cleaved by β-galactosi-dase, but it is not an inducer. Mutant cells unable to cleave this compound grow in its presence. Three types of mutants have this property: the desired repressing mutants as well as lacZ and lacY mutants. Both of the undesired mutant types were easily eliminated by requiring the mutants to grow on lactose. The selection scheme was successful, and mutants were found that contained elevated amounts of lac repressor. The isolation of the lacI overproducer was the first clearexample of the successful isolation of a promoter mutation and was itself a breakthrough. The resulting IQ (Q for quantity) mutation mapped at the beginning of the I gene, as expected for a promoter mutation, and generated a 10-fold increase in the level of repressor.