AMPK Methods and Protocols

13. Pellet glutathione agarose by centrifugation at 3,350gfor
    3 min. Wash glutathione agarose three times by resuspension in
    1 ml of glutathione agarose wash buffer, followed by centrifu-
    gation at 3,350gfor 3 min.


14. For elution, after the final centrifugation step, resuspend in
    glutathione agarose elution buffer (10μl buffer /10μl aga-
    rose), and place on rotating wheel at 4C for 5 min. Pellet
    glutathione agarose by centrifugation at 3,350gfor 3 min.
    Dispense AMPK elution into 50μl aliquots (seeNotes 14and
    15 ). Flash freeze aliquots in liquid N 2 for long-term storage at
    
    80 C.

4 Notes

1. We find that maintenance of cDNAs for AMPK subunits on
   individual expression plasmids provides a highly dynamic plat-
   form for rapid generation of mutants, truncations, and isoform
   combinations of intact heterotrimers. Theoretically, any mam-
   malian cell-compatible expression constructs can be used; how-
   ever, we find that yields significantly drop if all three plasmids
   share the same promoter. For this reason we prefer to use a
   plasmid for γ-subunit expression with a different promoter
   (Ad-MLP) to that used forα-andβ-subunit expression (CMV).


2. COS7 and HEK293 cell lines express endogenous AMPK
   (e.g., bothβ1orβ2[ 10]) that can associate with recombinant
   subunits to produce complexes of undetermined isoform com-
   position. In the event that final application is isoform sensitive
   (e.g., drug screening), or to exclude wild-type subunits if
   mutant AMPK is desired, it is necessary to preferentially isolate
   the relevant recombinant subunit under investigation. We have
   found this is conveniently achieved by placing distinct affinity
   tags on each subunit (α1/2: GST;β1/2: myc or FLAG;γ1/2/
   3: HA). We have yet to detect any discernible effect of these
   (mostly small) tags on AMPK regulation.


3. We have found that overall yield is largely determined by
   expression levels of the recombinantβ-subunit, which forms
   the stabilizing scaffold for the AMPK heterotrimer. Inclusion
   of a C-terminal affinity tag can have profound effects on
   β-subunit expression, for unknown reasons. In particular, we
   find that protein yield is increased three- to fourfold by expres-
   sing theβ-subunit as a FLAG fusion. Affinity tags must be
   incorporated at theβ-subunit C-terminus to avoid disrupting
   the N-terminal myristoylation consensus sequence.


4. We experience higher transfection efficiencies and subsequently
   greater protein yields using FuGENE HD transfection regent

Production in Mammalian Cells 167