- Induce protein expression with 100μM (final concentration)
of isopropyl β-D-thiogalactopyranoside (IPTG), reduce the
temperature to 18C, and grow the cells for an additional 18 h. - Harvest the cells and re-suspend them in 50 ml of cell lysis
buffer. - Sonicate the cell suspension on ice for 2–4 min at 50% of duty
cycle. - Remove the insoluble material by centrifugation at 29,000g
in a centrifuge for 30 min at 4C. - Load the supernatant onto a nickel affinity chromatography
column and wash with 10 column volumes of cell lysis buffer. - Elute the bound proteins using a linear gradient (10 column
volume) with elution buffer. - Pool fractions containing the AMPK subunits based on 10%
SDS-PAGE analysis and dialyze overnight in dialysis buffer. - For further purification, load the AMPK protein on a size
exclusion chromatography column. - Isolate the appropriate fractions corresponding to AMPK het-
erotrimer (~135 kDa MW), which occur at approximately
70 ml of elution volume (if using the Superdex 200 HiLoad
16/60 column) depending on injection volume and column
connections.
For expression of AMPK in insect cells, followsteps 15– 21 below.
- Custom synthesize the genes for human AMPK subunits for
expression in insect cells (seeNote 3). - Subclone them individually into theBamHIandXhoIsites
insect cell expression vector (pFastBac1) under the control of
polyhedrin promoter using standard molecular biology
techniques. - Generate recombinant viruses and amplify them (seeNote 14).
- For expression of the heterotrimeric complex, infect 1 l of Sf-9
cells at a cell density of 2 106 viable cells/ml and an MOI of
0.5 at 1:1:1 ratio ofα,β, andγsubunit containing viruses in a
serum-free insect cell medium (Fig.1b). - Grow the cells for 48–72 h after infection and harvest them
when their viability reaches 80–88%. - Store the harvested cells at 80 C until further use.
- For purification of AMPK, followsteps 7– 14 above.
3.2 Hydrogen/
Deuterium Exchange
Coupled to Mass
Spectrometry
Buffer exchange all recombinantly purified human AMPK hetero-
trimeric proteins (full-length or truncated) into protein storage
buffer and dilute to a final working concentration of 10μMin
H 2 O HDX buffer [11](seeNote 15).
Biophysical Studies to Evaluate Protein-Ligand Interactions 37