Establishing the affinity purification procedure

2.4.3.1 Purification from E. coli

The CBD was PCR-amplified from pWL-CBD using the primers PrR16CBD-fo and PrR16CBD-re. The PCR product was digested with NcoI and NdeI, and the CBD cloned into the respective sites of pET15. The resulting plasmid was verified by sequencing the manipulated regions and transformed toE. coli strain BL21.

Purification was done according to the following protocols:

Protocol Eco1 A preculture (25 ml LB with ampicillin) was grown overnight on a shaker (150 rpm) at 37◦C. 3 ml preculture were used to inoculate the main culture (100 mlLB Amp), which was grown under identical conditions. When an OD600of 0.6

was reached, the expression was induced by addition of IPTG to a final concentration of 1 mM. After4 hthe cells were pelleted by centrifugation at 4000 x g, 4◦Cfor20 min. They were resuspended in1 mlresuspension buffer (RB: 300 mMK Cl, 100 mMNa Cl, 2 mM Pi, 0.5 mM PMSF, pH 7.5), and sonified (6 x20 s, output control 5, duty cycle

50 %) on ice-water. The lysate was cleared by centrifugation (14000 rpm, 20 min,

4◦C), and the supernatant transfered to a fresh tube. 50µl 10 % (w/v) cellulose suspension (fibrous medium, Sigma) in RB were added and the mixture incubated for 1 h in an overhead shaker. The cellulose was spun down (3000 rpm, 3 min, RT), and the pellet washed with 200 µl RB. The cellulose was pelleted as before and the supernatant removed. The washing step was repeated three times. Now the pellet was resuspended in 40µl LDS sample buffer and the suspension boiled for 5 min at

100◦C. The tube was centrifuged (5000 rpm, 5 min) and the supernatant transfered to a fresh tube. 7.5µllysate and supernatant (after cellulose incubation) and 15µlof the washing and elution (boiling) fractions were loaded on a gel.

Protocol Eco2 Expression and lysate preparation were done as described above. A cellulose column was prepared by pipetting 350µl cellulose suspension (10 % (w/v) Sigma fibrous medium in RB) into a Mobicol empty spin column. The column was cen- trifuged (300 x g, 1 min, RT), washed with500µl RB to remove fines, and centrifuged again.

600 µllysate were applied to the column and the cellulose resuspended. After1 min

the column was centrifuged (2000 x g, 1 min), and the flowthrough discarded. This step was repeated with the rest of the lysate. The column was washed five times with

500µlCFE and centrifuged as before. For elution, 100µl ethylene glycol were added, the cellulose resuspended, and after 1 min incubation the column was centrifuged as before. The eluate was collected in a fresh reaction tube. The elution was repeated twice. 15µlof the washing and elution fractions were loaded on a gel.

Protocol Eco3 Essentially the same as protocol 2. The differences were the use of microcrystalline cellulose (Avicel PH-101, Fluka) as matrix, and the column was centrifuged only with 300 x g for 20 s in all centrifugation steps.

2.4.3.2 Purification from H. salinarum

Protocol Hsa1 The bait expression strain was precultured in35 mlHalomedium con- taining0.15µg/mlnovobiocin at37◦Con a shaker (150 rpm) until an OD600 of 0.5-1.0

was reached. 1 ml of this preculture was used to inoculate 100 ml Halomedium. The main culture was incubated on a shaker (110 rpm) at 37◦C. When the main culture had reached an OD600 of 0.6 to 1.0, cells were harvested by centrifugation (8000 rpm,

15 min, 8◦C) and resuspended in 1-2 ml CFE buffer (3 M K Cl, 1 M Na Cl, 400 mM

N H4Cl, 40 mM Mg Cl2, 10 mM Tris/H Cl, pH 7.5) + 0.01 % Triton X100 + 0.5 mM

PMSF. Cells were lysed by sonication on ice water (2 x20 s, Branson sonifier 250,3 mm

disruptor horn, output level 2, constant) and the lysate cleared by centrifugation at 14000 rpm, 4◦Cfor 20 min in a tabletop centrifuge.

In the meantime a cellulose column was prepared by pipetting300 µlcellulose sus- pension (10 % (w/v) Avicel PH-101 in CFE) into a Mobicol empty spin column. The column was centrifuged (300 x g,1 min, RT), washed with600µlCFE to remove fines, and centrifuged again.

The cleared lysate was applied to the column in 600µl portions and the cellulose resuspended by vortexing. After 1 min incubation at room temperature the column was centrifuged (300 x g,1 min, RT) and the flow-through discarded. The cellulose was washed twice with 600µl CFE. After each washing step the column was centrifuged (300 x g, 1 min, RT) and the flow-through discarded. An additional centrifugation (770 x g, 30 s, RT) was performed after the last washing step to reduce the amount of retained buffer. For elution, 200µl ethylene glycol were applied to the column, the cellulose resuspended, and the column centrifuged. The eluate was collected in a fresh microfuge tube and elution repeated. Proteins were precipitated with ice-cold acetone. 3µl lysate and flow-through, 15µl of the washing fraction, and the total eluted protein were loaded on a gel.

Protocol Hsa2 Similar to protocol Hsa1. The main culture had a volume of1 l, and the resulting cell pellet was resuspended in 3 ml CFE + 0.1 % Triton X100. Elution fractions were pooled.

Protocol Hsa3 Similar to protocol Hsa2. Elution was performed twice with 400µl

ethylene glycol.

Protocol Hsa4 A main culture of200 mlHalomedium was grown as described above. At an OD600 of around 1.0, cells were harvested by centrifugation (8000 rpm, 15 min,

15◦C) and resuspended in 1.6 ml CFE + 0.1 % Triton X100. Cells were lysed by sonication on ice water (2 x 20 s, Branson sonifier 250, 3 mm disruptor horn, output level 2, constant), and the lysate cleared by centrifugation at 14000 rpm, 18◦C for

20 minin a tabletop centrifuge. Cellulose columns were prepared as described before. The cleared lysate was applied to the column in 600 µl portions and the cellulose resuspended by pipetting up and down. After1 min incubation at room temperature the column was centrifuged (300 x g,1 min, RT) and the flow-through discarded. The cellulose was washed twice with600µlCFE + 0.1 % Triton X100 and once with CFE. After each washing step the column was centrifuged (300 x g,1 min, RT) and the flow- through discarded. An additional centrifugation (770 x g, 30 s, RT) was performed after the last washing step to reduce the amount of retained buffer. For elution,600 µl

ethylene glycol were applied to the column, the cellulose resuspended by pipetting up and down, and the column centrifuged. The eluate was collected in a fresh microfuge tube and proteins were precipitated with ice-cold acetone. The total eluted protein was loaded on a gel.