Expression and purification of FtsW and RodA from Streptococcus pneumoniae,two membrane proteins involved in cell division and cell growth,respectively |
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Authors: | Noirclerc-Savoye Marjolaine Morlot Cécile Gérard Philippe Vernet Thierry Zapun André |
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Affiliation: | Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale J.-P. Ebel (CEA/CNRS/UJF), 41 rue Jules Horowitz, 38027 Grenoble, France. |
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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. |
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