The M. tuberculosis raaS gene was cloned into the NdeI and NheI sites of the pET15-Tev plasmid to generate a hexahistidine-tagged recombinant protein (4 (link)). Protein expression in Escherichia coli BL21(DE3) was induced by isopropyl β-d-thiogalactopyranoside at a final concentration of 0.2 mm. Recombinant RaaS was purified using a HiTrap 1-ml IMAC HP column (Amersham Biosciences). Site-directed mutants of RaaS were generated using a GeneArt mutagenesis kit (Invitrogen) according to the manufacturer's instructions.
Turapov O., Waddell S.J., Burke B., Glenn S., Sarybaeva A.A., Tudo G., Labesse G., Young D.I., Young M., Andrew P.W., Butcher P.D., Cohen-Gonsaud M, & Mukamolova G.V. (2014). Oleoyl Coenzyme A Regulates Interaction of Transcriptional Regulator RaaS (Rv1219c) with DNA in Mycobacteria. The Journal of Biological Chemistry, 289(36), 25241-25249.
Corresponding Organization : University of Leicester
Other organizations :
Brighton and Sussex Medical School, University of Sussex, St George's, University of London, Centre de Biologie Structurale, Centre National de la Recherche Scientifique, Université de Montpellier, Inserm, Aberystwyth University, Institute of Biological, Environmental and Rural Sciences
Cloning of the M. tuberculosis raaS gene into the NdeI and NheI sites of the pET15-Tev plasmid
Generation of site-directed mutants of RaaS using a GeneArt mutagenesis kit
dependent variables
Recombinant RaaS protein expression in Escherichia coli BL21(DE3)
Purification of recombinant RaaS using a HiTrap 1-ml IMAC HP column
control variables
Concentration of isopropyl β-d-thiogalactopyranoside used to induce protein expression (0.2 mM)
controls
No positive or negative controls were explicitly mentioned in the given information.
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