2.6.1 PAS domains

Cell lysis

All PASp and PASc constructs were puried following the same protocol. Cell pellets from 1 L expression cultures were re-suspended in 60 mL lysis buer (20 mM Tris·HCl pH 7.9, 300 mM NaCl, 10 mM imidazole, 0.5 mM phenylmethylsulfonyluoride (PMSF)) on ice. Per 100 mL of lysis buer, one cOmplete (EDTA free) protease inhibitor cocktail tablet (Roche Diagnostics, Basel, CH) was added. Cells were ruptured by sonication, cellular debris was removed by centrifugation (45 minutes at 48,000 g, 4 C).

Ni-NTA purication

In a rst purication step, Ni-NTA (nitrilotriacetic acid) resin (Qiagen, Hilden, GER) was used to enrich histidine-tagged PAS domains. 2.5 mL resin were equilibrated with 50 mL lysis buer before adding the supernatant from cell lysis. The suspension was mixed on a tilting shaker for one hour at 4 C and transferred to a disposable plastic column (Thermo Fisher Scientific). The ow-through was collected and the resin washed with 100 mL lysis buer. Target proteins were eluted with 8×2 mL elution buer A (20 mM Tris·HCl pH 7.9, 300 mM NaCl, 100 mM imidazole) and 8 × 2 mL elution buer B (20 mM Tris·HCl pH 7.9, 300 mM NaCl, 500 mM imidazole). Purication was monitored with SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis).

Gels were run for 60 minutes at 120 V (see table 2.4). Protein fractions containing the target protein were pooled and dialysed over night in 2 L TEV-buer (50 mM Tris·HCl pH 7.9, 0.5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.5 mM PMSF) at 4 C.

2. Materials and Methods 25

Table 2.4: 17.5 % SDS-PAGE gels Stacking gel

30 % acrylamide 10 %

Tris·HCl pH 6.8 (1 M) 12.5 %

10 % sodium dodecylsulfate (SDS) 1 % 10 % ammonium persulfate (APS) 1 % tetramethylethylenediamine (TEMED) 0.08 %

Protein concentration of dialysed protein was approximated from UV-absorption spectra.

The N -terminal His-tag was cleaved by adding 1 mg TEV protease per 100 mg protein and incubating for six hours at room temperature without stirring. Successful cleavage was veried by SDS-PAGE. The protein sample was then added to 2.5 mL of Ni-NTA agarose (equilibrated with 50 mL SEC buer: 20 mM sodium phosphate pH 6.5, 150 mM NaCl) and incubated for one hour. The resin was washed with 10 mL SEC buer, ow-through and wash fractions were pooled and dialysed over night at 4 C against 2 L of SEC buer.

Size exclusion chromatography

The dialysed protein samples were concentrated to 1.5 mL using Vivaspin 20 (10 kDa) spin concentrators (Sartorius, Göttingen, GER). The protein concentration was monitored via UV absorption spectra. Concentrated samples were puried via size exclusion chro-matography (SEC) on a Superdex 75 16/60 column (GE Life Sciences, Little Chalfont,

26 2. Materials and Methods

UK). The column was equilibrated with two column volumes SEC buer, protein fractions were analysed by SDS-PAGE and fractions containing pure protein were pooled. The nal PAS domain samples were dialysed over night at 4 C against 2 L of NMR buer (20 mM sodium phosphate pH 6.5, 50 mM NaCl). The buer was used both for liquid-state NMR experiments and crystallisation set-ups. In a nal step, samples were concentrated to 1.4 - 1.8 mM using Vivaspin 20 (10 kDa molecular weight cut-o) concentrators and 0.01 % sodium azide was added. All samples were stored at 4 C when not used in experiments.

For determining the oligomeric state of proteins, analytical SEC was carried out on a Superdex 75 10/300 column (GE Life Sciences). 100 µL protein samples at con-centrations of 1 mM and the SEC buer described above were used. To calculate the sample molecular weight based on elution volume, a standard protein mix (Gel ltration calibration kit LMW, GE Life Sciences) was separated on the same column. 1.5 mg each of conalbumin, carbonic anhydrase, ribonuclease A and aprotinin were suspended in 500 µL SEC buer; 100 µL of this sample was used for SEC.

2.6.2 Gt CitApc solid-state NMR samples

Solid-state NMR samples were kindly provided by Karin Giller (Department of NMR-based structural biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, GER) using the following protocol.

Cell lysis

Cell pellets were re-suspended on ice in 225 mL TKMD buer (50 mM Tris·HCl pH 7.0, 200 mM KCl, 5 mM MgCl2, 5 mM β-mercaptoethanol, one spatula tip of DNAseI, two cOmplete (EDTA free) protease inhibitor cocktail tablets, 0.5 mM PMSF) using a Potter-Elvehjem homogeniser. The suspension was stirred at 4C for one hour before cell lysis utilising three cycles in a french pressure cell (20,000 psi). Cell debris was pelleted by centrifugation (30 minutes at 4 C and 27,500 g), the supernatant was spun down in an ultracentrifuge (90 minutes at 4C and 102,089 g). The ultracentrifugation pellet was re-suspended in 180 mL Ni-NTA-buer (20 mM Tris·HCl pH 7.9, 500 mM NaCl, 10 mM imidazole) with 5 mMβ-mercaptoethanol, two cOmplete (EDTA free) protease inhibitor

2. Materials and Methods 27

cocktail tablets, 0.5 mM PMSF and 4 % Triton X-100 (v/v) using a Potter-Elvehjem homogeniser.

The suspension was stirred for three hours at 4 C and subjected to ultracentrifugation a second time (one hour at 4 C and 109,251 g). The supernatant was diluted to an end volume of 900 mL using Ni-NTA-buer with three cOmplete (EDTA free) protease inhibitor cocktail tablets and 0.5 mM PMSF to set the nal concentration of Triton X-100 at 0.8 % (v/v).

Ni-NTA purication

The protein solution was loaded over-night onto a 5 mL Ni-NTA column (GE Life Sciences) at 0.7 mL/min and 4 C. The column was washed with Ni-NTA-buer (sup-plemented with half a tablet of cOmplete (EDTA free) protease inhibitor cocktail per 100 mL, 1 mM β-mercaptoethanol, 0.5 mM PMSF and 0.2 % (v/v) Triton X-100) at 3 mL/min until a baseline was reached. The detergent was changed on-column by wash-ing with 80 mL of Ni-NTA-buer supplemented with half a tablet of cOmplete (EDTA free) protease inhibitor cocktail per 100 mL, 1 mM β-mercaptoethanol, 0.5 mM PMSF and 1 % (v/v) laurydimethylamine-oxide (LDAO) at 3 mL/min.

One-step elution was carried out by washing with elution buer (20 mM Tris·HCl pH 7.9, 500 mM NaCl, 500 mM imidazole) supplemented with one tablet of cOmplete (EDTA free) protease inhibitor cocktail per 100 mL, 1 mM β-mercaptoethanol, 0.5 mM PMSF and 1 % (v/v) LDAO at 2 mL/min. 2 mL fractions were collected and analysed by SDS-PAGE.

Size exclusion chromatography (SEC)

Pure protein fractions were pooled and concentrated to 2.5 mL using Vivaspin 20 (30 kDa) spin concentrators. The protein concentration was monitored via UV absorption spectra.

Concentrated samples were puried via size exclusion chromatography on Superdex 200 26/60 columns (GE Life Sciences). The column was equilibrated with two column volumes gel ltration buer (20 mM Tris·HCl pH 7.4, 150 mM NaCl, 1 mM DTT, 0.3 % LDAO) and 2 mL fractions were collected. Protein fractions were analysed by SDS-PAGE

28 2. Materials and Methods

gel electrophoresis and pure protein was pooled.

Reconstitution in liposomes

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)- or asolectin lipid lms were set up by dissolving the appropriate amount of lipids for a protein/lipid molar ratio of 1:100 in 2 mL of chloroform and methanol (1:1). The solvent was removed in a nitrogen gas stream followed by lyophilisation over-night. The lipid lm was sonicated (3×15 minutes) in 1 mL gel ltration buer before adding ultraltered protein samples. The mixture was incubated for two hours at room temperature on a tilting shaker. LDAO was subsequently removed by incubation with 2 g Bio-Beads SM-2 (Bio-Rad Laboratories, Hercules, USA) per 66 mg LDAO on a tilting shaker at 4 C over night. The sample was diluted to 40 mL with gel ltration buer without LDAO and the Bio-Beads were removed on a disposable plastic column which was washed with 2×10 mL gel ltration buer without LDAO.

The liposomes were pelleted by ultracentrifugation (two hours at 4 C and 109,251 g) and the pellet washed and re-suspended with 60 mL of 20 mM Tris·HCl pH 7.4. Ultra-centrifugation was repeated (one hour at 4C and 109,251 g) and the pellet re-suspended in 10 mL of 20 mM Tris·HCl pH 7.4. After another ultracentrifugation step (one hour at 4C and 109,251 g), the pellet was re-suspended in 1 mL of 20 mM Tris·HCl pH 7.4. This step was repeated, following a nal centrifugation run (one hour at 4 C and 109,251 g) after which the supernatant was removed, leaving 20 µL of buer. The sample was spun down another 30 minutes at 4C and 109,251 g and stored at 4 C before transferring to solid-state NMR rotors.

For producing citrate-bound G. thermodenitricans CitApc R93A, the same protocol was used, adding 5 mM sodium citrate to every buer following cell lysis.

2. Materials and Methods 29

Im Dokument Transmembrane Signalling: Structural and Functional Studies on Histidine Kinase CitA (Seite 32-37)