Purification of bacteriophage lambda repressor

• The purification of lambda repressor using recently developed chromatography media is described.
• Ion exchange followed by heparin affinity was used to obtain lambda repressor in high purity.
• Lambda repressor looping and binding specificity was assayed using single molecule techniques.
• Lambda repressor looping and binding specificity was similar with or without a C-terminal His-tag.

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy “footprinting” assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops.