- Pass clarified lysate over the nickel column using a peristaltic
pump at 1 ml/min. - Wash the nickel column with lysis buffer until the concentra-
tion of protein eluting is low, approximately 10 CV. - Elute AMPK by passing 1–2 CV of elution buffer over the
nickel column, pool the protein peak, and concentrate to
5 ml if necessary. - Apply the protein peak to a SEC column, pre-equilibrated in
storage buffer. - Concentrate the AMPK containing fractions to ~5.0 ml for
CaMKK2 phosphorylation. - Phosphorylate AMPK by incubation with CaMKK2 at a ratio
of 1:2400 (w/w) (CaMKK2/AMPK) in the presence of
2.5 mM MgCl 2 , 0.5 mM ATP, and 0.5 mM AMP. Incubate
the phosphorylation reaction mixture at 22C for 1 h with
gentle rolling. - Terminate the reaction by applying the phosphorylation reac-
tion mixture to a SEC column (repeatstep 17). - Concentrate the pure AMPK containing fractions to ~10 mg/
ml, and flash freeze in liquid nitrogen. Store the purified
enzyme at 80 C. The yield from typical protein purification
is between 10 and 15 mg/L of culture.
3.2 Production
and Purification
of CaMKK2 for AMPK
Phosphorylation
- Culture the Sf21 insect cells in Sf-900 II media, incubating in a
shaking incubator at 26C with shaking at 150 rpm. - Infect the Sf21 cells at a multiplicity of infection of 10 and
continue incubation for 72 h post-infection. - Harvest the Sf21 cells by centrifugation at 1,000g for
15 min at 4C; discard the supernatant. - Wash the pellet with PBS and store at 80 C.
- Thaw and resuspend the Sf21 cells in 0.05culture volume of
ice-cold insect lysis buffer with protease inhibitors. Maintain at
4 C at all times. - Lyse Sf21 cells and clarify the lysate by centrifugation at
40,000 gfor 60 min at 4C; transfer supernatant to a
fresh tube. - Equilibrate FLAG monoclonal antibody-coupled affinity resin
in 10volumes of insect lysis buffer. - Add equilibrated FLAG monoclonal antibody-coupled affinity
resin to the Sf21 lysate, and mix on a rotating wheel at 4C for
1h.
22 Christopher G. Langendorf et al.