title: Expression and purification of FtsW and RodA from Streptococcus pneumoniae, two membrane proteins involved in cell division and cell growth, respectively.
abstract: FtsW and RodA are homologous integral membrane proteins involved in bacterial cell division and cell growth, respectively. Both proteins from Streptococcus pneumoniae were overexpressed in Escherichia coli as N-terminal His-tagged fusions. Their membrane addressing in E. coli was demonstrated by cell fractionation and was confirmed for FtsW by immunolocalization. Recombinant FtsW and RodA were solubilized from membranes using 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO). The detergent was exchanged to polyoxyethylene 8 lauryl ether (C12E8) during one-step purification procedure by Co(2+)-affinity chromatography. This procedure yielded 50-150 microg protein per litre of culture. Both proteins are likely to be folded as they are resistant to trypsin digestion and could be incorporated into reconstituted lipid vesicles.
authors.affiliation: Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale J.-P. Ebel (CEA/CNRS/UJF), 41 rue Jules Horowitz, 38027 Grenoble, France.
authors.forename: Marjolaine
authors.initials: M
authors.lastname: Noirclerc-Savoye
authors.forename: Cécile
authors.initials: C
authors.lastname: Morlot
authors.forename: Philippe
authors.initials: P
authors.lastname: Gérard
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
authors.forename: André
authors.initials: A
authors.lastname: Zapun
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002455:Cell Division; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D013296:Streptococcus pneumoniae / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; C061684:FtsW protein, Bacteria; D014357:Trypsin