Expression and purification of ArnR and ArnR1

Overexpression of ArnR from pSVA2543 and ArnR1 from pSVA2538 was performed essentially as described (Studier, 2014) using the E. coli BL21 (DE3) derivative strain E. coli OverExpress(tm)C43(DE3) (Lucigen). Cells were grown for approximately 48 h to OD₆₀₀ of 12. Membranes were isolated essentially as described (Bischof et al., 2016), using buffer A (20 mM Tris/HCl, pH 8, 300 mM NaCl). solubilization was performed for 2 h at 4 °C, using five mg/ml total membranes in solubilization buffer (buffer A supplemented with 10 mM Imidazole, 2% n-Dodecyl-β-d-maltopyranoside (DDM) (Glycon)). Solubilized protein was isolated from residual membranes using ultracentrifugation (400,000 × g, 4 °C, 1 h) and purified using one ml of His-Select nickel affinity gel resin (Sigma) equilibrated in solubilization buffer. Solubilized protein was applied to the resin and the flow-through was collected. After washing of the column with 5 × 1 ml of wash buffer (buffer A supplemented with 20 mM Imidazol, 0.5% DDM (Glycon)), bound target protein was eluted using 5 × 500 µl buffer A supplemented with 0.03% DDM and 150 mM Imidazole. Eluted ArnR protein was desalted to buffer A supplemented with 0.03% DDM using a PD10 column (GE Healthcare, Chicago, IL, USA), according to the manufacturer’s protocol. Eluted ArnR1 protein was further purified using a Heparin-HP column (GE Healthcare, Chicago, IL, USA) on an Äkta purifier system (GE Healthcare, Chicago, IL, USA). Therefore, fractions containing ArnR1 were diluted to 20 mM NaCl in buffer A supplemented with 0.03% DDM and loaded on a five ml HisTrap HP at one ml/min flow rate. After washing of the column with 20 mM Tris/HCl pH 8, 20 mM NaCl, 0.03% DDM for at least five column volumes, ArnR1 was eluted in two ml fractions using 20 mM Tris/HCl, pH 8, 500 mM NaCl, 0.03% DDM. ArnR as well as ArnR1 was concentrated using ultrafiltration in a 100 kDa cut-off Amicon (Merck Millipore, Burlington, MA, USA). Protein concentration was determined using BCA assay (Serva), according to the manufacturer’s protocol. The Analysis of the oligomeric state of purified ArnR and ArnR1 was performed using Blue-Native PAGE analyses as described (Claeys, Geering & Meyer, 2005) using NativeMark™ unstained protein standards (Life Technologies, Inc., Carlsbad, CA, USA).

In vitro phosphorylation assays of ArnR and ArnR1 with γ[³²P]-ATP (Hartmann Analytic) were performed as described (20). The reaction buffer contained 20 mM Tris/HCl pH 8, 150 mM NaCl. In a total final volume of 15 µl, two µM kinase and three µM ArnR or ArnR1 were mixed and 0.8 mM non-radioactive ATP and 0.3 mM γ[³²P]-ATP were added to the samples.

To show that phosphorylation of the kinases was specific and further show that ArnR and ArnR1 do not possess auto-phosphorylation activity negative control samples were taken along, that contained only the respective protein and the ATP mixture. In another negative control, the kinases were incubated in the absence of ATP and ArnR and ArnR1. After incubation of all samples at 55 °C for 10 min, 5× SDS-loading dye was added to a final concentration of 1 times to stop the reaction. Proteins were separated on 11% SDS gels and exposed on a phosphostorage screen (Molecular Dynamics, Sunnyvale, CA, USA) overnight. Screens were scanned using Typhoon FLA 7000 (GE Healthcare, Chicago, IL, USA). Thee independent experiments were performed and a representative phosphoimage is shown.