aureus. Staphylococcus aureus N315 (Ito et al., 1999) was used as a template for PCR amplification of the stk1, sa0077 and sarA
genes. Escherichia coli DH5α strain was used to propagate plasmids in cloning experiments. Escherichia coli BL21 (DE3) and E. coli BL21 (DE3) AD494 were used for expression experiments. The plasmid vector pET15 was purchased from Novagen. Escherichia coli strains were grown in Luria–Bertani (LB) medium at 37 °C. For strains carrying drug resistance genes, ampicillin and kanamycin were added to the medium at a concentration of 100 and 25 μg mL−1, respectively. Total DNA from S. aureus N315 served as a template in PCR amplification for preparing the stk1, sa0077 and sarA genes with appropriate restriction sites at both ends (Table 1). Each DNA fragment synthesized was restricted by appropriate GSK2126458 solubility dmso enzymes, and then ligated into the pET15b Obeticholic Acid vector opened with the same enzymes. The resulting plasmids were termed pET15b-sa0077, pET15b-stk1 and pET15b-sarA. In each case, the nucleotide sequence of the amplified gene was checked by dideoxynucleotide sequencing (Sanger et al., 1977). Escherichia coli BL21 (DE3) competent cells were transformed with plasmids pET-sar and pET-stk1. Cells from this strain were used to inoculate 1 L of LB medium supplemented with
ampicillin and were incubated at 37 °C under shaking until the A600 nm reached 0.5. Isopropyl-β-d-thiogalactopyranoside (IPTG) was then added at a final concentration of 1 mM. After 6 h, His6-SarA and His6-Stk1 were extracted and purified using an immobilized Zn2+ matrix (Qiagen), suitable for the purification of fusion proteins carrying a poly-histidine tag. The production of the His6-SarA protein was confirmed by analysis of Coomassie blue-stained polyacrylamide gels. Protein concentration was determined using the Coomassie Plus Protein Assay (Pierce). For His6-SA0077 overexpression, E. coli AD494 cells were transformed with the appropriate pET15b-sa0077 plasmid and grown under the same conditions as above. SA0077 was not soluble and was retained
in inclusion bodies. It was therefore extracted after a step of denaturation/renaturation. Briefly, the pellet obtained was resuspended in buffer B (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 20% w/v Orotidine 5′-phosphate decarboxylase sucrose), then centrifuged for 10 min at 6000 g, incubated for 25 min in iced water and centrifuged again for 10 min at 8000 g to obtain spheroplasts that were resuspended in 10 mL buffer C [1 × phosphate-buffered saline (PBS), 5 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride (PMSF)]. After sonication, DNAse I and RNAse A were added at a final concentration of 60 μg mL−1 each. The preparation was then centrifuged for 30 min at 10 000 g and the pellet was washed twice with buffer D (1 × PBS, 5 mM EDTA, 1% Triton X-100). Solubilization of inclusion bodies was achieved by treatment of the pellet with 10 mL buffer E (50 mM Tris-HCl, pH 8.0, 6 M guanidinium chloride, 25 mM dithiothreitol (DTT), 5 mM EDTA) incubated for 1 h on ice.