Expression of recombinant proteins

2.2.9.1 Preparation for induction

Plasmids with fusion protein constructs (GST-ADAM27, MBP-DCE, MBP-CE, and MBP-D) isolated from DH5α cells were transformed to expression host bacterial strain E.coli BL21 (DE3). Single colony of bacteria containing vector with fusion construct was picked from a freshly streaked plate and 50 ml LB culture with kanamycin / ampicillin was inoculated.

2.2.9.2 Sample induction protocol

1. Bacterial culture was incubated with shaking at 37°C until OD600 reached 0.4–1.

2. Uninduced sample was removed for the control (see TCP sample protocol). To the remainder, IPTG from a 100 mM stock was added to a final concentration of 0.4 mM and incubation was continued for 2–3 hrs.

3. Induced sample was removed (see TCP sample protocol).Flasks were placed on ice for 5 min and then cells were harvested by centrifugation at 5000 x g for 5 min at 4°C.

4. Cells were resuspended in 0.25 culture volume of cold 20 mM Tris-HCl pH 8.0, and centrifuged as above.

5. Finally supernatant was removed and cells were stored as a frozen pellet at –70°C or used directly for purification according to BugBuster method or using amylose resin.

2.2.9.3 Analysis of protein from bacterial cultures.

2.2.9.3.1 Total cell protein (TCP) sample

The expression of target genes was assessed by analysis of total cell protein on a SDS-polyacrylamide gel followed by Coomassie blue staining.

1. Prior to harvesting the cells, 1 ml sample of well-mixed culture was taken and centrifuged at 10,000 x g for 1 min.

2. Pellet was resuspended by mixing in 150 µl of 1X phosphate-buffered saline (PBS).

3. 50 µl of 4X sample buffer and fresh DTT was added and sample was sonicated for 20 sec.

4. Proteins were heated for 10 min at 70°C to denature and then stored at –20°C until SDS-PAGE analysis.

5. When required, detection of the expressed protein was achieved by Western blotting.

2.2.9.3.2 Preparation of cell extracts with BugBuster™ protein extraction reagent

a. Soluble fraction

1. Bacteria were harvested from liquid culture by centrifugation at 6,500 x g for 5 min.

Pellet was drained to remove as much liquid as possible.

2. Cell pellet was resuspended in the BugBuster at room temperature, using 2 ml reagent for cells from a 50 ml culture.

3. Cell suspension was incubated on a shaking platform at a slow setting for 10 min at room temperature.

4. Insoluble cell debris was removed by centrifugation at 16,000 x g for 20 min at 4°C.

Pellet was saved for inclusion body purification as described below.

5. Supernatant - soluble extract was transferred to a fresh tube.

6. Sample was subjected to SDS-PAGE electrophoresis.

b. Inclusion body purification

1. Induced culture was processed according to steps 1–4 above for the soluble protein fraction.

2. Pellet from step 4 above was resuspended in the same volume of BugBuster reagent that was used to suspend the cell pellet. Pellet material was vortexed to obtain an even suspension.

3. Lysozyme was added to a final concentration of 200 µg/ml. Sample was mixed by vortexing and incubated at room temperature for 5 min.

4. 6 volumes of 1:10 diluted BugBuster reagent (in deionized water) was added to the suspension and vortexed for 1 min.

5. Suspension was centrifuged at 16,000 x g for 15 min at 4°C to collect the inclusion bodies. Supernatant was removed.

6. Inclusion bodies were resuspended in ½ the original culture volume of 1:10 diluted BugBuster, mixed by vortexing, and centrifuged as in step 5. Wash step was repeated 2 more times.

7. The final pellet of purified inclusion bodies was resuspended in 1X PBS.

8. Sample was subjected to SDS-PAGE electrophoresis.

2.2.9.4 Purification of MBP fusion proteins using amylose resin.

1. 1 liter LB + glucose and ampicillin was inoculated with 10 ml of an overnight culture of cells containing the fusion plasmid.

2. Bacterial culture was induced as described above.

3. Cells were harvested by centrifugation at 4000 x g for 20 min. Cell pellet was resuspended in 400 ml 30 mM Tris-HCl, 20% sucrose, pH 8.0. EDTA was added to 1 mM and sample was incubated for 5–10 min at room temperature with shaking.

4. After centrifugation at 8000 x g for 20 min at 4°C, supernatant was removed; pellet was resuspended in 400 ml of ice-cold 5 mM MgSO4 and shaken for 10 min in ice bath.

5. Sample was centrifuged at 8000 x g for 20 min at 4°C. Collected supernatant was the cold osmotic shock fluid.

6. 8 ml of 1 M Tris-HCl, pH 7.4, was added to the osmotic shock fluid.

7. Amylose resin was poured in a 2.5 x 10 cm column. Column was washed with 8 volumes of column buffer.

8. Osmotic shock fluid from step 6 was loaded at a flow rate of about 1 ml/min.

9. Column was washed with 12 column volumes of column buffer.

10. Fusion protein was eluted with column buffer + 10 mM maltose. 10 to 20 fractions of 3 ml each were collected (fraction size = 1/5th column volume). The fraction with fusion protein was detected by Bradford protein assay.

11. The protein-containing fractions were pooled and concentrated using Amicon Centricon concentrator.

12. Sample was subjected to SDS-PAGE electrophoresis.

2.2.9.5 Reduction of recombinant proteins.

MBP-CE and -DCE recombinant proteins are rich in cystein residues and during expression in bacteria intermolecular disulfide bonds could be formed resulting in large multimeric aggregates. It was described (Evans et al., 1997) that nonreduced multimers did not bind to eggs; in order to have egg-binding activity of recombinant proteins, reduction protocol was necessary.

To prepare protein for egg-binding studies, recombinant ADAM27-CE, and -DCE was reduced by treating with 20 mM DTT for 30–60 min at room temperature. The sample was then dialyzed extensively against a Hepes-buffered Whitten’s medium compatible dialysis buffer (WHITCO; 109.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4 , 0.23 mM sodium pyruvate, 4.8 mM sodium lactate, 7mMNaHCO3, 15mMHepes, 0.1 mg/ml gentamicin). The protein was then concentrated to 1 mg/ml; protein concentration was determined using Bradford method.