Expression and purification of FtsW and RodA from Streptococcus pneumoniae, two membrane proteins involved in cell division and cell growth, respectively.
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.
Noirclerc-Savoye
Marjolaine
M
Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale J.-P. Ebel (CEA/CNRS/UJF), 41 rue Jules Horowitz, 38027 Grenoble, France.
Morlot
Cécile
C
Gérard
Philippe
P
Vernet
Thierry
T
Zapun
André
A
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
D001426:Bacterial Proteins; D008565:Membrane Proteins; C061684:FtsW protein, Bacteria; D014357:Trypsin