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

Abstract

A dimeric inert form of the replication initiator RepE of the F plasmid was expressed, purified and crystallized in complex with the repE operator DNA. Diffraction data were collected to 3.14 Å resolution. 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 Å after the application of dehydration and crystal annealing and belong to space group P2{sub 1}, with unit-cell parameters a = 60.73, b = 99.32, c = 95.00 Å, β = 108.55°.

Authors:
 [1];  [2];  [1];  [3]
  1. Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)
  2. Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8315 (Japan)
  3. (Japan)
Publication Date:
OSTI Identifier:
22360311
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 4; Other Information: PMCID: PMC2330221; PMID: 17401213; PUBLISHER-ID: pu5174; OAI: oai:pubmedcentral.nih.gov:2330221; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ANNEALING; CRYSTALS; DEHYDRATION; DIFFRACTION; DIFFUSION; DIMERS; DNA; ESCHERICHIA COLI; HISTIDINE; MONOMERS; PROTEINS; RESOLUTION; SALTS; SPACE GROUPS

Citation Formats

Nakamura, Akira, Wada, Chieko, Miki, Kunio, E-mail: miki@kuchem.kyoto-u.ac.jp, and RIKEN SPring-8 Center at Harima Institute, Koto 1-1-1, Sayo, Hyogo 679-5148. Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107012894.
Nakamura, Akira, Wada, Chieko, Miki, Kunio, E-mail: miki@kuchem.kyoto-u.ac.jp, & RIKEN SPring-8 Center at Harima Institute, Koto 1-1-1, Sayo, Hyogo 679-5148. Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA. United Kingdom. doi:10.1107/S1744309107012894.
Nakamura, Akira, Wada, Chieko, Miki, Kunio, E-mail: miki@kuchem.kyoto-u.ac.jp, and RIKEN SPring-8 Center at Harima Institute, Koto 1-1-1, Sayo, Hyogo 679-5148. Sun . "Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA". United Kingdom. doi:10.1107/S1744309107012894.
@article{osti_22360311,
title = {Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA},
author = {Nakamura, Akira and Wada, Chieko and Miki, Kunio, E-mail: miki@kuchem.kyoto-u.ac.jp and RIKEN SPring-8 Center at Harima Institute, Koto 1-1-1, Sayo, Hyogo 679-5148},
abstractNote = {A dimeric inert form of the replication initiator RepE of the F plasmid was expressed, purified and crystallized in complex with the repE operator DNA. Diffraction data were collected to 3.14 Å resolution. 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 Å after the application of dehydration and crystal annealing and belong to space group P2{sub 1}, with unit-cell parameters a = 60.73, b = 99.32, c = 95.00 Å, β = 108.55°.},
doi = {10.1107/S1744309107012894},
journal = {Acta Crystallographica. Section F},
number = Pt 4,
volume = 63,
place = {United Kingdom},
year = {Sun Apr 01 00:00:00 EDT 2007},
month = {Sun Apr 01 00:00:00 EDT 2007}
}