RESUMO
Lactose repressor (LacI) is one of the best studied prokaryotic transcriptional regulatory proteins till date. Detailed structural, biochemical and genetic studies are being carried out on LacI since four decades to understand its ligand binding properties and the basis of allosteric response. We applied directed evolution methods on LacI to generate mutants with altered allosteric properties. After testing several hosts and expression vectors, a robust expression and screening system was optimised for identifying LacI variants with altered allosteric properties. After two rounds of error prone PCR (polymerase chain reaction) and shuffling, four mutants were selected from several thousand mutants, for their ability to induce reporter gene expression at 1 microM of isopropyl beta-D-1-thiogalactopyranoside (IPTG). The observed combination of mutations in these four improved LacIs was not reported earlier. The mutant Lac repressors seem to operate as very good molecular switches by inducing gene expression at 1 microM of IPTG and confer 2-10 times higher level of gene expression as compared with the WT (wild -type).
