Heterologous expression and purification of proton pumps

2.3.1 Purification of E. coli wild type ATP-Synthase (F0F1)

Wild type E. coli ATP-Synthase (F0F1) was purified on the base of a slightly modified protocol published previously (Moriyama et al., 1991). The DK8-strain carrying pBWU13 (DK8/pBWU13) was kindly donated by Dr. Y. Moriyama (Dep. of Pharmacology, University of Okayama).

For the expression of the F0F1-complex a single colony from a Amp-LB plate was picked from a freshly streaked out glycerol-stock (stored at –80°C) and cultivated for ~8 hours in

~5 ml liquid LB (Luria-Bertani medium) (Sambrook et al., 2001), containing 100 µg ampicillin. One liter of minimal medium (Moriyama et al., 1991) containing 0.5 % glycerol (the sole carbon source), but no ampicillin, was then inoculated with the preculture (1:500) and grown at 37°C until an OD₆₀₀ of ~1-1.5 was reached (~24 hours).

The cells were then harvested by centrifugation and resuspended in 10 ml of 0.5 M sucrose, 50 mM Tris-Cl, pH 8.0. 50 mg lysozyme, 1µg/ml Leupeptin, 1µg/ml Pepstatin and 1 mg/ml DNase I were added and the suspension was incubated for 1.5 hours at 30°C.

Then EDTA (pH 8.0) was added to 5 mM and the suspension was incubated for additional 30 min. Then the suspension was diluted 10fold with water containing 1µg/ml Leupeptin, 1µg/ml Pepstatin, 1 mM PMSF and 5 mM dithiothreitol (DTT). Afterwards the suspension was tip-sonicated (Branson S250A) for 5 x 30s at an output of 3-4 and 30% duty cycle.

Then glycerol was added to 10% and the suspension was centrifuged at 4500 rpm at 4°C in a Beckmann JA-20 rotor. The turbid supernatant was collected and spun at 50,000 rpm in a Beckmann Ti 70 rotor for 45 min at 4°C. The brown opaque pellet was resuspended in 1 ml of 10 mM Tris-Cl pH 8, 5mM DTT, 2mM MgCl2, 10% glycerol and proteinase

inhibitors (1µg/ml Leupeptin, 1µg/ml Pepstatin). The membranes were snap frozen in liquid N₂ and stored at –80°C.

For the purification of F₀F₁ the membranes were thawed and washed in in 10 mM Tris-Cl pH 8, 5mM DTT, 2mM MgCl₂, proteinase inhibitors (1µg/ml Leupeptin, 1µg/ml Pepstatin) and 0.8 % n--octylglucoside (OG, final concentration from 20 % OG-stock, added just prior to the next step) in a final volume of 6 ml. The membranes were pelleted immediately in a Beckman 100.4-rotor with 70,000 rpm for 30 min at 4°C. Then the membranes were resuspended in 10 mM Tris-Cl, 2 mM MgCl2 , 5 mM DTT, 2% OG and proteinase inhibitors as above with a small potter homogenizer for incubation on ice for 30 minutes. The lysate was spun again at 70,000 rpm for 30 min at 4°C. Afterwards the supernatant was subjected to a glycerol density gradient (in 10mM Tris-Cl pH 8, 5 mM DTT, 2 mM MgCl2 5-30% glycerol,1 % OG) in a Beckmann SW55 rotor with (52,000 rpm (330,000 g Rmax) for 5 hrs at 4°C. For this, maximal 0.5 ml of supernatant was placed on top of 4.5 ml step-gradient. The gradient was prepared in a cold room with glycerol contents of 30, 25, 20, 15, 10 and 5% glycerol. Fractions were collected from bottom to top with a peristaltic pump and a glass capillary in sizes of ~300 µl. After analysis with SDS-PAGE the fractions enriched in F0F1 holoenzyme were pooled, snap-frozen in liquid N2

and stored at -80°C.

2.3.2 Purification of Bacillus sp. PS3 thermophilc ATP-Synthase (TF₀F₁)

ATP synthase holoenzyme (TF₀F₁) from the thermophilic Bacillus sp. PS3 was constitutively expressed in the E.coli strain DK8 (native unc operon deleted) from the plasmid pTR19ASDS (Suzuki et al., 2002). The -subunit, which is present in three copies per complex is N-terminally hexa-His-tagged allowing affinity purification. The cells were grown to an OD600 of 1.5-2 in the presence of 100µg ampicillin per ml in 2 L TB (terrific broth) (Sambrook et al., 2001) and harvested by centrifugation. The pellets were resuspended in 50 mM Tris-Cl (pH 8.0), 0.5 mM EDTA and 1mg/ml lysozyme and incubated at 37 °C under gentle shaking for 1 hour. MgCl2 was then added to a final concentration of 5mM and the suspension was sonicated 2 min on ice. Subsequently, DNaseI, Na2SO4 and sodium cholate were added to reach final concentrations of 1µg/ml, 250mM and 0.7% (wt/vol), respectively. The suspension was stirred for 20 min at 25°C

MATERIALS AND METHODS

and the spun at 20,000 g at 4°C. After this washing step, the pellet was resuspended in 100mM KCl, 20mM Imidazole, 5 mM MgCl2 and 1% n-dodecyl--D-maltopyranoside (DDM) (wt/vol), pH 7.6 stirred for 45 min at 25 °C and then subjected to centrifugation with 20,000g at 4°C. The resulting supernatant was batch incubated with ~ 2-3 ml Talon beads (Clontech) to capture TF0F1 via its tagged -subunit. The beads were washed with 10 column volumes of 100mM KCl, 20 mM Imidiazole, 5 mM MgCl₂ and 0.08% DDM at pH 7.6. Elution of the complex was achieved with 250mM Imidazole, 50 mM KCl, 5 mM MgCl2, 0.05 % DDM. The complex could be used already with the purity achieved, but was purified further except for some initial test experiments. For this the eluate from the IMAC (immobilized metal affinity chromatography) step was dialyzed against 20mM NaCl, 20mM Hepes-Na and 5 mM MgCl2, pH 7.5 for 3 hours at 25°C or overnight at 4°C.

After ultrafiltration through a 0.22 µm membrane, the sample was injected onto a MonoQ anion exchange column (GE, 5/50) operated on an ÄKTA-System (ÄKTA explorer, GE) at 4°C and eluted with a step gradient (4 column volumes (CV) 0-18% Buffer B, 3 CV 18-60

% Buffer B, 11 CV 60-100 % Buffer B) with Buffers A (20 mM NaCl, 20mM Hepes-Na, 5 mM MgCl2, 0.05 % DDM, pH 7.5) and B (1 M NaCl, 20mM Hepes-Na, 5 mM MgCl2, 0.05 % DDM, pH 7.5). The eluent flow was 1ml/min for 18 CV. Fractions were collected along the gradient between 18% and 65 % B in 300µl steps. This concentrated the sample about 10-fold. The peak fractions were pooled and subjected to a final polishing step (size exclusion chromatography, SEC) to remove eventually occuring F₀ or F₁ domains from the F₀F₁ complex. About 600-800 µl were injected onto a Superdex 200 HC 10/30 SEC-column (GE) and eluted with 100 mM KCl, 10mM Hepes-K, 5 mM MgCl₂, 0.05 % DDM, pH 7.4 at a flow of 0.3ml/min for 1.5 CV at 4°C. Fractions of 0.5 ml were collected. After analysis on SDS-PAGE the TF₀F₁ containing fractions were pooled, protein concentrations were measured and the complex was stored at 4 °C. Yields up to 10 mg purified TF₀F₁ from 2L culture were reached.