AMPK Methods and Protocols

2 Materials

2.1 Expression of
Recombinant AMPK

Expression constructs required for the method are described below

(seeNotes 1and 2 ). All plasmids were prepared by standard mini-

prep kit and stored (
 20 C) in nuclease-free H 2 O at concentra-

tions>200 ng/μl.

1. pDEST27-GST fusion human AMPKα1orα2. These plasmids
   are Gateway destination vectors that allow constitutive expres-
   sion of N-terminally GST-tagged human AMPK subunitsα 1
   (UniProt Q13131) orα2 (P54646). Transcription is under the
   control of the human cytomegalovirus (CMV) immediate-early
   promoter/enhancer. pDEST27 plasmids were generated by
   Gateway recombination technology (Thermo Life Sciences).


2. pcDNA3(-) human AMPK β1orβ2. These plasmids are
   derived from pcDNA3(-). Transcription is under the control
   of the CMV promoter. cDNAs for human AMPK subunitsβ 1
   (Q9Y478) orβ2 (O43741) were inserted into the cloning site
   using restriction sites XhoI and HindIII. Addition of FLAG or
   myc affinity tags at the C-terminus, if required, was made
   possible by incorporating the relevant sequence into the reverse
   primer used for PCR amplification (seeNote 3).


3. pMT2 AMPK humanγ1,γ2, orγ3. These plasmids are derived
   from expression vector pMT2, in which transcription is
   directed by the adenovirus major late promoter (Ad-MLP)
   [9]. cDNAs for human AMPK subunits γ1 (P54619),

Table 1
Comparison of posttranslational modifications present on recombinant AMPK generated by
commonly used expression systems. Basal phosphorylation (p) at major sites onα- andβ-subunits,
andβ-subunit myristoylation, are presented as approximate % stoichiometries, derived from intact
protein mass spectrometry or immunoblot analyses [8]. Co-expression of AMPK in bacteria with
N-myristoyl transferase (NMT) results in fully myristoylated species (
NMT vs.þNMT); expression of
β-G2A mutant in mammalian cells abrogatesβ-subunit myristoylation (WT vs. G2A). n.d., not
determined

AMPK subunit Modification

Recombinant expression system

Bacterial Insect Sf21 Mammalian

α1/2 pThr-172 None ~10% ~10%

pSer485 >70% n.d. >70%

β1/2 Myristoylation None (
NMT) ~50% 100% (WT)

100% (þNMT) None (G2A)

pSer108 >60% n.d. ~10%

pSer182 None n.d. >80%

Production in Mammalian Cells 161