AMPK Methods and Protocols

6. 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.


7. Harvest the cells and re-suspend them in 50 ml of cell lysis
   buffer.


8. Sonicate the cell suspension on ice for 2–4 min at 50% of duty
   cycle.


9. Remove the insoluble material by centrifugation at 29,000
   g
   in a centrifuge for 30 min at 4C.


10. Load the supernatant onto a nickel affinity chromatography
    column and wash with 10 column volumes of cell lysis buffer.


11. Elute the bound proteins using a linear gradient (10 column
    volume) with elution buffer.


12. Pool fractions containing the AMPK subunits based on 10%
    SDS-PAGE analysis and dialyze overnight in dialysis buffer.


13. For further purification, load the AMPK protein on a size
    exclusion chromatography column.


14. 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.

15. Custom synthesize the genes for human AMPK subunits for
    expression in insect cells (seeNote 3).


16. Subclone them individually into theBamHIandXhoIsites
    insect cell expression vector (pFastBac1) under the control of
    polyhedrin promoter using standard molecular biology
    techniques.


17. Generate recombinant viruses and amplify them (seeNote 14).


18. 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).


19. Grow the cells for 48–72 h after infection and harvest them
    when their viability reaches 80–88%.


20. Store the harvested cells at 80 C until further use.


21. 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