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1. Day 1:
MexB from Pseudomonas aeruginosa is encoded by pFB101. The MexB gene was amplified from P. aeruginosa genomic DNA and inserted in the NdeI and XhoI restriction sites of the pET30b+ vector. The construct contains a C-terminal hexahistidine tag.
- The plasmid is used to transform E. coli strain C43(DE3) 12, and the transformants are plated on LB agar containing 30 ug/mL kanamycin.
2. Day 2: Overnight Cultures:
- In the evening, 4 X 3 mL LB cultures containing 30 ug/mL kanamycin are inoculated from the fresh transformant colonies. Alternatively, the cultures can be inoculated from a frozen perm.
These small cultures are grown on a roller at 37°C overnight.
3. Day 3: Growing 6 Liter Cultures:
- In the morning, use the overnight cultures to inoculate 150 mL LB containing 30 ug/mL kanamycin. Grow the culture at 37° C on a shaker.
- In the afternoon, use the small culture to inoculate 6 x 1L 2XYT media containing 30 ug/mL kanamycin in Fernbach flasks. (Use 25 mL per culture for a 1:40 dilution). Grow the cultures at 37°C until they reach an OD600 of 0.4-0.6, about 1.5 hours
- When the cultures reach the proper density, induce protein expression by adding 0.5 mL 1M IPTG. Put all the flasks back in the shaker and continue to grow them at 30°C overnight.
4. Day 4: Harvesting Cells and Purifying the Protein:
- Add protease inhibitors, DNAse, and lysozyme to the buffer solutions as follows: To 50 mL of cell resuspension buffer, add 10 mg DNaseI (0.1 mg/mL final concentration), 1 Complete EDTA-free protease inhibitor tablet, and a pinch of lysozyme. To 60 mL of membrane resuspension buffer, add 1 protease inhibitor tablet. To another 50 mL of membrane resuspension buffer, add 1 protease inhibitor tablet. Keep all three solutions on ice.
- Centrifuge the cultures 30 min 5,000 rpm in large-scale centrifuge to harvest the cells.
- Resuspend the cells in 100 mL cell resuspension buffer (50 mM NaP, pH 7.0, 300 mM NaCl, 2 mM MgCl2, 1 Complete EDTA-free protease inhibitor tablet, 0.1 mg/mL DNAse I, pinch of lysozyme)
- Pass the cell solution twice through a French pressure cell at 12,000 psi (762 gauge pressure). Collect the cell lysate in a bottle kept cold on ice.
- Transfer the cell lysate to SS34 centrifuge tubes and centrifuge to remove cell debris for 30 min at 10,000 rpm at 4°C in an SS-34 rotor.
- Carefully remove the supernatant into Ti647.5 ultracentrifuge tubes. Centrifuge 50 min at 40,000 rpm at 4°C. Discard the supernatant.
- Resuspend the pellet, which contains the cell membranes, in approx. 25 mL of membrane resuspension buffer (50 mM NaP, pH 7.0, 300 mM NaCl, 5% glycerol, 1 Complete EDTA-free protease inhibitor tablet).
- Transfer the membrane suspension to a clean centrifuge tube and centrifuge at 40,000 rpm in a Ti647.5 rotor for 50 min at 4 °C.
- Discard the supernatant and resuspend the washed membrane pellet in 25 mL membrane resuspension buffer (50 mM NaP, pH 7.0, 300 mM NaCl, 5% glycerol, 1 Complete EDTA-free protease inhibitor tablet).
5. TM Protein Solublization:
- To the resuspended membranes (about 25 mL), add 6 mL 10% DDM (final detergent concentration = 2% DDM) Rock the mixture at 4 °C for 2 hours.
- Centrifuge the mixture at 40,000 rpm for 40 min at 4°C in the Ti647.5 rotor to separate the soluble protein detergent complexes from the insoluble proteins. Save the supernatant, which contains the MexB protein detergent complexes.
6. IMAC:
- Mix the supernatant obtained from the high speed spin with the talon metal affinity beads equilibrated in resuspension buffer. Incubate for 1hr on a roller at 4°C.
- Pour the slurry into a gravity flow column body and discard the flow through.
- Wash the column with 20 mL (10 column volumes) of IMAC Binding and Wash Buffer (50 mM NaP, pH 7, 300 mM NaCl, 5% glycerol, 0.2% DDM)
- Elute the protein with IMAC elution buffer (50 mM NaP, pH 7.0, 300 mM NaCl, 5% glycerol, 250 mM imidazole, 0.2% DDM).
- Take 15 μL samples of the elution fractions, mix each with 15 μL 2X SDS sample buffer for analysis by polyacrylamide gel electrophoresis. Spin 30sec in a microcentrifuge. Analyze the samples on a 10% polyacrylamide SDS gel to estimate the amount and purity of MexB in each fraction.
- Pool the fractions containing the MexB protein detergent complexes and concentrate them in a spin concentrator at 4° C. Be careful that the protein does not precipitate out at this step.
7. Gel Filtration Column:
- Pre-equilibrate a Superose 12 HL 30/10 column with 24 mL running buffer (50 mM NaP, pH 7.0, 300 mM NaCl, 5% glycerol, 0.2% beta-octylglucoside), and wait for a flat baseline.
- Rinse the Akta system loading loop with running buffer.
- Filter the protein solution using a syringe filter before applying it to the column.
- Load up to 240 μL of the protein solution onto the column, with a protein concentration of up to 5mg/ mL.
- Run 1.5 column volumes (36 mL) of buffer, collect 0.25 mL fractions. The MexB protein detergent complexes should elute as a peak at around 10 - 15 mL of elution volume.
- Take 5 μL samples of the peak fractions. Mix each sample 1:1 with 2X SDS sample buffer. Analyze the samples on a 10% polyacrylamide gel to estimate the amount and purity of MexB in each fraction
- Pool the fractions containing pure MexB.
8. Representative Results:
Figure 1 includes a polyacrylamide gel with pooled column fractions from the IMAC column and individual fractions from the gel filtration column. After the gel filtration column the protein appears pure by Coomassie stained polyacrylamide gel. Figure 2 includes a trace from the gel filtration column showing the main peak of the protein detergent complex eluting from the column. The average yield of MexB protein is approximately 2 mg per 6 liters of 2XYT culture.
Figure 1. SDS-PAGE gel of purification of MexB PDCs. Lane 1, Molecular weight markers. 2, Pooled IMAC fractions. 3-7, Gel filtration column fractions.
Figure 2. Example of Gel Filtration Results for MexB protein detergent complexes (PDC).