Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA.
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.
Nakamura
Akira
A
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Wada
Chieko
C
Miki
Kunio
K
D001483:Base Sequence; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074164:RepE protein, E coli; D012097:Repressor Proteins