Application Notes

6 detection of very low protein concentrations in the range of a few µg/ml in as little as 10 µl of sample, thus protein consumption is very small compared to other methods. Last, since no equilibration times or washing steps are needed, more than 400 thermal unfolding experiments can be performed per day, thus guaranteeing fast and efficient screening of a variety of buffer conditions. Material and Methods Protein preparation HiTehA was cloned into a modified pET28b vector using NcoI and NotI restriction site and protein was expressed with a C-terminal 3 x flag – 10 x His-tag in the E. coli strain C43 (DE3). Bacteria were grown in Terrific Broth Media (TB) in the presence of 50 mg l -1 kanamycin to an absorption at 600 nm of 0.6-0.8 at 37 °C. Then protein expression was induced with 0.4 mM isopropyl-β-D- thiogalactopyranoside (IPTG) and continued for 4 hours at 37 °C. Cells were centrifuged and lysed by sonication in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, protease inhibitor Set III (1:1000 final dilution). Bacterial membranes were obtained by ultracentrifugation and membrane proteins were extracted with 1 % (w/v) DDM (Carl Roth) over night under mild agitation at 8 °C. The supernatant of an ultracentrifugation step (150,000 g, 35 min, 4 °C) was loaded onto a Nickel-affinity-column (1 ml HisTrap Excel column, GE Healthcare). The column was washed with 20 CVs of 25 mM and 50 mM imidazole respectively. HiTehA was eluted with a gradient of 50 to 500 mM imidazole ranging over 20 CVs. Further protein purification was performed employing size exclusion chromatography using a Superdex 200 (XK 16/60, GE Healthcare) column and 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.02 % DDM as running buffer. HiTehA-containing fractions were pooled and concentrated up to 1.2 mg/ml using a VivaSpin6 ultrafiltration devices (100 kDa cut off). (Modified purification protocol after Chen et al., 2010) In order to promote detergent exchange, DDM- solubilized HiTehA (in 50 mM Tris HCl pH 8, 200 mM NaCl and 0.02 % DDM) as obtained from the above-described preparation, was first diluted into a 10 x concentrated Tris-buffer indicated in Table 1. Selected detergents from the Memb- PASS™ Differential Filtration Detergent Screen from Jena Bioscience (Jena, Germany) were used. After 30 minute incubation at RT, 20 µl of the protein were applied to Pierce Detergent Removal Spin Column (0.5 ml), and prepared following the manufacturers protocol, except that the protein was directly eluted into 20 µl of a 2 x concentrated Tris buffer (Table 1). Thermal stability screen by nanoDSF HiTehA solutions were loaded into nanoDSF grade capillaries which were then loaded into the Prometheus NT.48. Each sample was measured in duplicate. Unfolding of HiTehA was detected during heating in a linear thermal ramp (1 °C/min, 25-90 °C) at low detector sensitivity and with an excitation power of 10 %, and unfolding transition points were determined from changes in the emission wavelengths of tryptophan fluorescence at 350 and 330 nm. Unfolding transition points were automatically identified by the Prometheus NT. Control software. Unfolding onset temperatures were determined after baseline correction and normalization of the data. The unfolding onset was defined as the point at which 1 % of protein was unfolded. To evaluate the concentration dependence of HiTehA unfolding, a 7 fold serial dilution of the protein in 50 mM Tris HCl pH 8, 200 mM NaCl and 0.02 % DDM was prepared, starting at a concentration of 120 µg/ml. Differential scanning calorimetry DSC experiments were carried out using a NanoDSC device (TA Instruments). 300 µl of a solution of 1.6 mg/ml HiTehA protein in 10 mM Sodiumphosphate, 67 mM Sodiumsulfate, 0.02 % DDM, pH 8 was used. 30 µl of the same solution were used for triplicate Tm determination using the Prometheus NT.48. Prior to experiments, the protein solution was dialyzed against the buffer for 24 hours, and buffer as well as protein solution were degassed. Before starting thermal unfolding experiments, 3 equilibration cycles including heating from 10 °C to 100 °C and subsequent cooling to 10 °C were carried out. Thermal unfolding of HiTehA was recorded after a 5 minute equilibration time at 10 °C using a thermal ramp from 10 °C to 100 °C with a heating rate of 1 °C/min at a constant pressure of 3 bar. Buffer alone served as reference. After finishing the DSC experiments, the device was cleaned by subsequently flushing the cells with ddH 2 O, 1 % SDS, ddH 2 O, 1 M NaOH, and ddH 2 O.

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