Protein purification

Ultrasonic disintegration: Cells, suspended in the appropriate buffer, were filled into a glass Rosetta cell, kept on ice and broken by ultrasonication on a Branson 250 Sonifier (Heinemann, Germany). The duty time was 5 minutes at 50% duty cycle. The process was repeated several times.

French press: The suspended cells were filled into a bottle. The cell suspension was sucked into a pre-cooled French press cell (American Instruments, Maryland, USA) and the cells were disrupted by applying a pressure of 110 MPa. The broken cells were collected into the bottle and refilled into the pressure cell for another cycle of disrupting. The cycle was repeated 3  4 times and the cells were observed under a microscope to verify optimal cell disruption.

3.2.2 Determination of protein concentration Bradford method

Protein concentration was determined by the Bradford method (Bradford, 1976). The assay is based on the shift of the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 from 465 nm to 595 nm upon binding of protein. Standards with 0 – 1 mg of BSA were made up to 10 µl and 200 µl Coomassie Brilliant G-250 reagent. The reactions were incubated in the dark at room temperature for 30 minutes and the absorbance was measured at 595 nm by a microplate spectrophotometer.

44 Bicinchoninic acid (BC) assay

The BC assay (Uptima-interchim, France) is a colorimetric assay: it involves the reduction of Cu²⁺ to Cu⁺ by peptidic bounds of proteins (Smith et al, 1985). The bicinchoninic acid chelates Cu⁺ ions with very high specificity to form a water soluble purple colored complex.

This assay particularly suits to analyze complex mixtures containing nucleic acids, lipids or detergents (Triton X-100, SDS extracts). Therefore, the assay was used to determine the concentration of membrane proteins.

3.2.3 Polyacrylamide gel electrophoresis (PAGE) SDS-PAGE

The samples were mixed with SDS sample buffer (125 mM Tris/HCl pH 6.8, 10% glycerol, 10% mercaptoethanol, 4% SDS, 0.2% bromophenol blue) in the ratio of 1:1 and boiled at 95 °C for 5 minutes to denature the proteins. Boilng step was avoided in case of preparation of membrane proteins. The running buffer was 25 mM Tris pH 8.8, 190 mM glycine, 0.1%

SDS. Electrophoresis was run at constant voltage of 200 mV until the bromophenolblue marker reached the end of the gel. The proteins were stained by heating the gel with 0.1%

Coomassie Brilliant Blue R-250 in methanol/water/glacial acetic acid (4:5:1) shortly and leaving it at room temperature for 10 minutes. The gel was distained by heating it with ethanol/water/glacial acetic acid (4:5:1) and incubating overnight on a shaker.

SDS-PAGE gel (15% acrylamide)

Stock solution Separating gel (µl) Stacking gel (l)

1M Tris/HCl, pH 8.8 2250 -

1M Tris/HCl, pH 6.8 - 353

H2O 315 1905

10% SDS 63 30

Acrylamide/Bisacrylamide

(40%/1.6%) 2780 712

5% TEMED 63 30

10% Ammonium peroxodisulfate 90 60

45 Native PAGE

For molecular mass determination of native enzymes, a 4  20% gradient gel (Mini-PROTEAN^TGX^TM Precase Gels, BIO-RAD), various concentrations of acylamide gels, and a marker (SERVANative Marker Liquid Mix for BN/CN, SERVA Electrophoresis) were used.

The protein samples were mixed with 2  sample buffer for clear native electrophoresis (SERVA) in the ratio of 1:1. The running buffer was 25 mM Tris, 192 mM glycine, pH 8.3.

Electrophoresis was run at voltage of 100 mV and constant current of 0.01 A until the bromophenolblue marker reached the end of the gel. Staining and destaining steps were as same as SDS-PAGE (3.2.3).

Contents for various concentrations of acrylamide gels

10% (µl) 9% (µl) 8% (µl) 6% (µl)

1.5 M Tris/HCl, pH 8.8 1880 1880 1880 1880

H2O 3100 3350 3600 4100

Acrylamide/Bisacrylamide (40%/1.6%) 2500 2250 2000 1500

100% TEMED 5 5 5 5

10% Ammonium peroxodisulfate 25 25 25 25

3.2.4 Preparation of soluble membrane protein

The 5 g wet packed S. aciditrophicus cells were suspended in 50 mM phosphate buffer pH 7.4 and the cells were broken by three passages through French press cell at 110 MPa. Cell debris was removed by centrifugation at 10,000  g for 20 min at 4 °C. The crude extract was centrifuged at 120,000  g for 1 h. The membrane extract was collected and washed twice with 50 mM phosphate, pH 7.4 by centrifuging at 120,000  g for 30 minutes. The washed membrane extract was solubilised with 20 mM phosphate pH 7.4 containing 0.5 M NaCl, 10 mM EDTA and 2% n-dodecyl-ß-D-maltoside (DM) and homogenized well to solubilise it further. The suspension was on ice for 30 minutes. The solubilised membrane was centrifuged at 120,000  g for 30 minutes and the solubilised membrane extact in the supernatant was collected (Buckel, 1986a).

46 3.2.5 Purification of recombinant Re-citrate synthase from S. aciditrophicus

Recombinant Re-citrate synthase from S. aciditrophicus was purified aerobically using a Strep Tactin column at 4 °C. The harvested E. coli cells were suspended in equilibration buffer and opened using sonication. Cell debris was removed by ultra-centrifigation at 100,000  g for 1 h. The clear supernatant was filtrated and loaded on the affinity Strep Tactin column, which was equilibrated with equilibration buffer. After loading the cell free extract, the column was washed at least 10 column volumes of equilibration buffer. To release the co-purifying chaperone from the target protein, a simple one step MgCl2/ATP/KCl incubation procedure was applied. Incubation of the target protein immobilized on the Strep Tactin resin with 10 mM MgCl2/10 mM ATP/150 mM KCl for 2 h at 4 °C. The protein was eluted with equilibration buffer with 2.5 mM D-desthiobiotin. At last, the purified protein from Strep-Tactin column was loaded on a gel filtration column (HiLoad^TM26/60 Superdex^TM200) to achieve the pure protein.

Buffers for recombinant protein purification;

Equilibration buffer 50 mM K-phosphate, pH7.4, 75 mM NaCl

Dissociation buffer 20 mM HEPES/NaOH, pH 7.0, 10 mM MgCl₂, 10 mM ATP, 150 mM NaCl

Elution buffer 50 mM Tris/HCl pH, 8.0, 150 mM NaCl, 2.5 mM desthiobiotin Gel filtration buffer 50 mM Tris/HCl pH, 8.0, 150 mM NaCl

3.2.6 Purification of glutaconyl-CoA decarboxylase from S. aciditrophicus

All purification procedure was carried out under aerobic conditions at 4 °C. The membrane extracts were prepared as mentioned earlier (3.2.4). The solubilised membrane was loaded onto SofLink^TM Soft Release Avidin (Promega, USA) column (1  10 cm) equilibrated with the buffer (20 mM phosphate, pH 7.0, 0.1% DM). The column was washed with 10 column volume of equilibration buffer at a rate of 0.2 ml per minute until there was no protein detected. The protein was eluted with elution buffer (20 mM phosphate, pH 7.0, 0.1% DM, 2 mM D-biotin). The enzyme containing fractions were pooled and concentrated by ultrafiltration over a Centricon (cut off size: 1 kDa) and stored at − 80 °C (Buckel, 1986a).

47 3.2.7 Purification of the subunits of recombinant glutaconyl-CoA decarboxylase from S. aciditrophicus

GcdA by Strep-tag

Wet packed cells were suspended in 100 mM Tris/HCl, pH 8.0, 150 mM NaCl and lysed by three times of French press cell at 110 Mpa. The cell free extract was prepared after centrifugation for 1 h at 100,000  g. The supernatant was filtrated and loaded on the Strep Tactin column. After washing with 5 column volumes of wash buffer (100 mM Tris/HCl, pH 8.0 and 150 mM NaCl), the protein was eluted by elution buffer (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin).

GcdB by His-tag

The membrane extracts were prepared as mentioned earlier. The solubilised membrane was load onto the Ni-Sepharose column. After washing with 5 column volumes of wash buffer (50 mM phosphate, pH 7.4, 0.5 M NaCl, 20 mM imidazole, 0.1% DM), the protein was eluted by buffer containing 50 mM phosphate, pH 7.4, 0.5 M NaCl, 500 mM imidazole containing DM.

GcdAC by affinity SofLink^TM Soft Release Avidin column

GcdAC was purified by SofLink^TM Soft Release Avidin column. Purification was performed by routine procedure mentioned above.

3.2.8 Partial purification of recombinant glutaconate CoA-transferase from A.

fermentans

Cells of E. coli BL21(DE3), harboring the plasmid with glutaconate CoA-transferase from A.

fermentans, were suspended in 15 ml of 20 mM potassium phosphate, pH 7.4 and sonicated for 15 minutes (divided in three intervals) with a Branson sonifier. Cell debris was removed by ultra-centrifugation at 100,000  g for 1 h at 4 °C. Ammonium sulphate was added to the cell free extract to achieve 50% saturation. After centrifugation at 25,000  g for 30 minutes, the supernatant was brought to 80% ammonium sulphate saturation and centrifuged as described above. The protein pellet was dissolved in 40 ml of 20 mM potassium phosphate

48 buffer, pH 7.4 and partially purified with Centricon cut-off membrane of 100 kDa. The purified protein was stored at 4 °C (Mack et al, 1994).

3.2.9 Gel filtration

To separate the Re-citrate synthase from co-purified chaperone, the protein solution was loaded on a HiLoad^TM26/60 Superdex^TM200 column which was prior washed by H₂O and equilibrated by 50 mM Tris/HCl, pH 8.0. The chromatography was achieved using 50 mM Tris/HCl, pH 8.0 and 150 mM NaCl with a flow rate of 2 ml/min.