Purification and characterization of truncated human AMPK α2β2γ3 heterotrimer from baculovirus-infected insect cells

AMP-activated protein kinase (AMPK) is considered an important target for treatment of type II diabetes and the metabolic syndrome. The muscle-specific isoform of the regulatory γ-subunit, γ3, within the context of AMPK α2β2γ3 complex, is involved in glucose and fat metabolism in skeletal muscle. In an effort to identify γ3-specific activators of AMPK, we have produced truncated human recombinant AMPK α2β2γ3 (huα2β2γ3trunc) for biochemical characterization. Infection of insect cells with three baculoviral stocks encoding the individual subunits resulted in soluble expression of a stable huα2β2γ3trunc heterotrimeric complex. Co-expression of the three subunits was essential for solubility since the individual protein components, when expressed separately, were almost completely insoluble. The huα2β2γ3trunc heterotrimer was purified to apparent homogeneity from baculovirus-infected insect cells in a 1:1:1 stoichiometric complex. The huα2β2γ3trunc heterotrimer had significant circular dichroism signal that was lost as a function of temperature, implying that the purified protein was folded. The huα2β2γ3trunc complex was capable of binding AMP and ATP, although the heterotrimer showed preference for AMP, in particular, as seen by thermal denaturation circular dichroism analyses. Further experiments showed that the truncated complex bound ZMP (AICAR-monophosphate) albeit with much lower affinity than AMP. To investigate whether there were isoform-specific differences in the nucleotide binding affinities, a well-characterized truncated mammalian α1β2γ1 (mα1β2γ1trunc) equivalent of huα2β2γ3trunc was also purified. The γ1 and γ3 isoforms showed comparable nucleotide binding affinities and solution behavior properties.