Preparation of Membrane Scaffold Proteins

MSP protein expression and purification

Materials

• 50 mg Kanamycin

• 1 M IPTG stock (2.383 g in 10 mL)

• 1 M imidazole (68.077 g in 1 L)

• 1 M Tris-HCl, pH 8.0 (121.14 g in 1 L)

• 5 M NaCl (146.1 in 500 mL)

• 0.5 M EDTA (93.05 g in 500 mL)

Procedure

Expression

1. Grow BL21 (DE3) cells at 37C in 4-L LB Kana medium until OD600 = 0.7.

2. Induce with 1 mM IPTG (final conc.).

3. Harvest after shaking for 3-4 hours at 37C.

4. Centrifuge at 5000xg for collecting the cells.

Purification

1. Resuspend frozen cell pellet in 50 mM Tris-Cl pH 8.0 500 mM NaCl, 1 mM EDTA, protease inhibitor cocktail. (10 mL/gram)

2. Break cells – French press.

3. Centrifuge in F20-12 × 50 LEX rotor (at 30000 xg, 30 min, 4C).

4. Apply supernatant onto Ni-NTA column (~1 mL resin/1 L cell culture) equilibrated with 50 mM Tris pH 8.0, 500 mM NaCl, 1% TX-100 (50x CMC). (Buffer A: 50 mM Tris-HCl pH 8.0)

5. Wash with 5 CV Buffer A + 1% TX-100; Wash with 5 CV Buffer A + 50 mM Cholate; Wash with 5 CV Buffer A, no detergent; Wash with 5 CV Buffer A + 20 mM imidazole; Elute with 3 CV Buffer A + 500 mM imidazole.

6. Measure the protein concentration (Extinction coefficient = 19.94)

7. Add TEV protease (1 A280 TEV for 20 A280 MSP protein). Add DTT to final concentration to 1mM. Dialysis against buffer (50mM Tris-HCl pH8.0, 500mM NaCl, 1mM DTT, 10% Glycerol). Incubate overnight at 4C.

8. Apply onto NiNTA, wash with 20mM imidazole, and collect flow-through and wash fractions. Equilibrate w/ 5 CV Buffer A; Wash w/ 5CV Buffer A + 20 mM imidazole; Elute w/ 3CV Buffer A + 500 mM imidazole.

9. Gel filtration chromatography using Superdex 200. (Running buffer: 20 mM Tris-HCl pH 8.0, 100 mM NaCl)

10. Concentrate up to 1 mM, flash freeze in liquid nitrogen, and store at -80C.