AMPK Methods and Protocols

into several distinct stages, each broadly consisting of 3–4 days of

laboratory work. In terms of hours worked in the laboratory and

scheduling of equipment, the most intensive is the purification of

AMPK from a pellet of bacteria which have expressed the protein.

The pellet can be frozen before use. However, the purification

process after thawing the pellet is a 3-day process that in our

hands has been most effectively carried out in a continuous process.

The protocol below is based on growth of 10 L of bacterial

culture from 122 L flasks each containing 800 mL. This starting

volume typically yields 50 mg of purified AMPK protein, which can

be safely stored frozen at around 5–10 mg/mL in 10–20μL ali-

quots at 80 C until use for crystallization.

3.2 Expression
and Production
of AMPK in E. coli
(4-Day Protocol)

Carry out all procedures at room temperature unless otherwise

specified.

Day 1:

1. Make up LB agar plates containing 100μg/mL ampicillin (two
   per transformation plus one for each flask of cells you wish to
   grow (in this example, for 12 flasks make 14 plates)).


2. Add 1μL (50–1000 ng) of plasmid DNA (full-length AMPK,
   γ1[human, 1–331], AVI-tag-β1[human, 1–270], His-α 1
   [human, 1–552] in tricistronic vector) to one aliquot of
   BL21 Star (DE3)E. colicompetent cells.


3. Incubate for 10 min on ice.


4. Transfer to a 42C heat block or water bath for 45 s to heat
   shock.


5. Immediately return the cells to ice for 2 min.


6. Allow cells to recover in 250μL SOC media at 37C for 1 h.


7. Spread two different volumes (10 and 50μL) of recovered cell
   mixture onto two LB-agar-ampicillin plates.


8. Incubate the plates overnight at 37C in a microbiological
   incubator.
   Day 2:


9. First thing in the morning, pick a few colonies from the trans-
   formation plates and grow in 10 mL LB broth 100μg/mL
   ampicillin in 50 mL plastic tubes at 37C with shaking at
   200 rpm until the OD at 600 nm (1 cm path length cuvette)
   is between 2 and 3.


10. Take one culture that has grown (discard the rest). Plate out
    200 μL of culture onto one LB-agar-ampicillin plate for every
    flask you wish to grow (e.g., if you are going to make 12 flasks,
    plate out 12 plates with 200μL each). Use 10μL inoculation
    loops for streaking out cultures and 1μL inoculation loops for
    picking clones. Incubate plates overnight at 37C.

AMPK Crystallisation 5