- 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. - 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
- 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). - 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. - 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. - We experience higher transfection efficiencies and subsequently
greater protein yields using FuGENE HD transfection regent
Production in Mammalian Cells 167