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Rajamohan, Francis; Harris, Melissa S.; Frisbie, Richard K.; Hoth, Lise R.; Geoghegan, Kieran F.; Valentine, James J.; Reyes, Allan R.; Landro, James A.; Qiu, Xiayang; Kurumbail, Ravi G.
Protein expression and purification, 10/2010, Volume: 73, Issue: 2Journal Article
AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (α) subunit and two regulatory (β and γ) subunits. The muscle-specific AMPK heterotrimeric complex (α2β2γ3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK α2β2γ3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK α2β2γ3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK α2β2γ3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the α 2 domain. The un-assembled β 2 and γ 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK α2β2γ3 complex was 1.1 mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKβ and LKB1. The kinase activity of activated AMPK α2β2γ3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK α2β2γ3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.
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