Preparation of XaxAB pore complexes

Protein Expression

Transformation of competent E. coli cells

1. Thaw competent E. coli BL21 RIPL (DE3) cells on ice for 10 minutes
2. Add 1–3 μl 100 ng of plasmid DNA to the cell mixture
3. Place the mixture on ice for 30 minutes
4. Heat shock at 42 °C for 90 seconds
5. Place on ice for 5 minutes
6. Add 800 μl of SOC media
7. Incubate for 60 minutes at 37 °C  in a thermocycler at 750 rpm
8. Plate 50-100 μl cells on agar plates containing 125 μg/ml ampicillin O/N

Expression Culture 

1. Inoculate 5 ml preculture of LB medium containing 125 μg/ml ampicillin with one colony of transformed cells
2. Incubate for 8 h at 37 °C
3. Inoculate 200 ml of LB medium containing 125 μg/ml ampicillin with 100 µl of preculture and incubate O/N at 37 °C
4. Inoculate 2 l of LB medium containing 125 μg/ml ampicillin with the preculture to an optical density (OD) of 0.1
5. Incubate at 37 °C until an OD600 between 0.5 – 0.8 is reached
6. Add 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG)
7. Incubate O/N at 20 ˚C
8. Harvest cells at 6000 rpm for 10 min 
9. Store pellet at −20 °C until use

Purification

1. Resuspend and lyse cells in lysis buffer (50 mM HEPES 7.5, 300 mM NaCl)
2. Press lysate 3-4 times under high pressure through a microfluidizer (lysate should be almost clear)
3. Centrifuge for 30 min at 38000 rpm and 4 °C 
4. Equilibrate a 5 ml His-Trap HP column (GE Healthcare Biosciences) with two column volumes of the IMAC column buffer (50 mM HEPES 7.5, 300 mM NaCl) containing 10 mM imidazole
5. Add 10 mM imidazole to the crude lysate 
6. Load crude lysate to the His-Trap HP column using a peristaltic pump

Next steps were carried out at an ÄKTApurifier system (GE Healthcare Biosciences)

1. Wash Ni-column with IMAC column buffer containing 10 mM imidazole
2. Elute bound Xax protein using an imidazole gradient from 10 mM up to 500 mM within 45 minutes
3. Dialyze fractions containing the protein O/N at 4 °C against IMAC column buffer (MWCO 12-14 kDa)
4. Load concentrated sample on HiLoad 26/60 Superdex 200 pg column (alternatively if available use Superose 6 for XaxA) 
5. Elute protein from the column with SEC buffer (20 mM HEPES pH 7.5, 200 mM NaCl)
6. Monomeric protein of XaxA (45 kDa) and XaxB (39 kDa) elute from the HiLoad 26/60 Superdex 200 pg at ~200 ml 

• Note: For analytical preps on Superose 6 or Superdex 10/300 GL columns, the monomeric proteins elute at ~16-17 ml)

Preparation of XaxAB pore complexes 

1. Mix equimolar concentrations (e.g. ~20 µM) of XaxA and XaxB 
2. Add 0.1 % cymal-6 of a 5 % cymal-6 stock solution (Note: Oligomers should form immediately at RT or 37˚C)
3. Recommended incubation times for pore formation
   1. 37 ˚C: ~5 minutes
   2. RT: ~30 minutes
   3. 4 ˚C: O/N
4. Add fivefold molar excess of amphipols A8-35 (Antrace) to the mixture
5. Incubate mixture at RT for 20 minutes
6. Remove detergent by dialysis against the SEC buffer (20 mM HEPES pH 7.5, 200 mM NaCl) O/N at RT
7. Isolate XaxAB pore complexes by SEC on a Superose 6 10/300 GL column (GE Healthcare Life Sciences)

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