Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA

Abstract

The replication initiator factor RepE of the F plasmid in Escherichia coli is an essential protein that stringently regulates the F-plasmid copy number. The RepE protein has a dual function: its monomer functions as a replication initiator, while its dimer acts as a transcriptional repressor of the repE gene. The wild-type dimeric RepE protein was expressed as an N-terminal histidine-tagged protein, purified under native conditions with a high salt concentration and crystallized in complex with the repE operator DNA using the sitting-drop vapour-diffusion technique. The crystals diffracted to a resolution of 3.14 A after the application of dehydration and crystal annealing and belong to space group P2(1), with unit-cell parameters a = 60.73, b = 99.32, c = 95.00 A, beta = 108.55 degrees.

Figure 1

DNA duplexes used in complex formation and crystallization trials. The common 8 bp sequences between iteron and operator are underlined and their directions are indicated by the arrows.

Figure 2

Native PAGE of the crystals of the His₆-RepE–DNA1 complex. Samples were loaded onto a 15% polyacrylamide gel (37.5:1 acrylamide:bisacrylamide) and electrophoresed. The gels were stained with either Mupid-Blue (left) or CBB (right) for the detection of DNA1 and His₆-RepE, respectively. The arrows labelled Complex and Free DNA indicate the positions of the His₆-RepE–DNA1 complex and free DNA1, respectively. Lane 1, solution used in crystallization; lane 2, crystallization mother liquor; lanes 3–4, washing solution; lane 5, dissolved crystals.

Figure 3

Crystal of the His₆-RepE–DNA1 complex. The scale bar is 100 µm in length.

Figure 4

Pseudo-precession image of a His₆-RepE–DNA1 crystal representing the h0l zone of reciprocal space. The circle indicates a resolution of 4 Å. The picture was generated with HKLVIEW (Collaborative Computational Project, Number 4, 1994 ▶).