3 Methods
3.1 Expression
and Purification
of AMPK
for Crystallization
- 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. - Heat shock cells by incubating at 42C for 45 s, then rest on
ice for 5 min. - Add 0.1 ml of LB to the transformation mixture and incubate
at 37C for 1 h. - Plate the transformation mixture onto LB agar plates contain-
ing 1/1000 ampicillin 1000stock and 1/1000 kanamycin
1000 stock, and incubate at 37C overnight. - Transfer a single colony into 3 ml of LB with 3μl of ampicillin
1000 stock and 3μl kanamycin 1000stock, and incubate at
37 C overnight. - 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). - Starter culture: In the expression flask, add 100 ml LB, 50μlof
AMPK glycerol stock, 100μl ampicillin 1000stock, and
100 μl kanamycin 1000stock. Incubate the starter culture
overnight in a shaking incubator at 37C with shaking at
120 rpm. - Expression culture: Add 900 ml of LB (supplemented with
900 μl ampicillin 1000stock 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 ). - Induce protein expression by adding 1.0 ml IPTG 1000stock
to the culture, and incubate in a shaking incubator at 16C
with shaking at 120 rpm overnight (approximately 20 h). - Pellet cultured cells by centrifugation at 3000gfor 20 min
at 4C and remove supernatant. - Resuspend pelleted cells in chilled lysis buffer (with 1000
protease inhibitor stock added), and maintain at 4C at all
times. - Lyse cells via cell disruption and then clarify lysate by centrifu-
gation at 44,000gfor 30 min at 4C; transfer supernatant
to a fresh tube. - 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