AMPK Methods and Protocols

3 Methods

3.1 Expression
and Purification
of AMPK
for Crystallization

1. Transformation of expression vectors: Add 50 ng of either
   AMPKα 2 β 1 γ1 or AMPKα1/α2 RIM chimera dual expression
   constructs into 20μl of Rosetta™2 (DE3)E. colicompetent
   cells, and incubate on ice for 30 min.


2. Heat shock cells by incubating at 42C for 45 s, then rest on
   ice for 5 min.


3. Add 0.1 ml of LB to the transformation mixture and incubate
   at 37C for 1 h.


4. Plate the transformation mixture onto LB agar plates contain-
   ing 1/1000 ampicillin 1000
   stock and 1/1000 kanamycin
   1000
   stock, and incubate at 37C overnight.


5. Transfer a single colony into 3 ml of LB with 3μl of ampicillin
   1000
   stock and 3μl kanamycin 1000
   stock, and incubate at
   37 C overnight.


6. Make a glycerol stock from the overnight culture. Add 100μl
   of autoclaved glycerol to 900μl of LB and store at 80 C(see
   Note 3).


7. Starter culture: In the expression flask, add 100 ml LB, 50μlof
   AMPK glycerol stock, 100μl ampicillin 1000
   stock, and
   100 μl kanamycin 1000
   stock. Incubate the starter culture
   overnight in a shaking incubator at 37C with shaking at
   120 rpm.


8. Expression culture: Add 900 ml of LB (supplemented with
   900 μl ampicillin 1000
   stock and 900μl kanamycin 1000
   
   stock) directly to the starter culture, and incubate in a shaking
   incubator at 37 C with shaking at 120 rpm until OD 600
   reaches 3.0 (seeNotes 4and 5 ).


9. Induce protein expression by adding 1.0 ml IPTG 1000
   stock
   to the culture, and incubate in a shaking incubator at 16C
   with shaking at 120 rpm overnight (approximately 20 h).


10. Pellet cultured cells by centrifugation at 3000
    gfor 20 min
    at 4C and remove supernatant.


11. Resuspend pelleted cells in chilled lysis buffer (with 1000
    
    protease inhibitor stock added), and maintain at 4C at all
    times.


12. Lyse cells via cell disruption and then clarify lysate by centrifu-
    gation at 44,000
    gfor 30 min at 4C; transfer supernatant
    to a fresh tube.


13. Equilibrate the nickel column with lysis buffer. Ensure to wash
    with at least 10 column volumes (CV) of buffer (CV¼5ml
    bed volume).

Visualization of Drug Binding Sites 21