Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase.
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 (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) 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 alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 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 alpha 2 domain. The un-assembled beta 2 and gamma 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 alpha2beta2gamma3 complex was 1.1mg/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 CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 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 alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.
Rajamohan
Francis
F
Pfizer Global Research and Development, Groton, CT 06340, USA. francis.rajamohan@pfizer.com
Harris
Melissa S
MS
Frisbie
Richard K
RK
Hoth
Lise R
LR
Geoghegan
Kieran F
KF
Valentine
James J
JJ
Reyes
Allan R
AR
Landro
James A
JA
Qiu
Xiayang
X
Kurumbail
Ravi G
RG
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000249:Adenosine Monophosphate / Q000378:metabolism; D001713:Biphenyl Compounds; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020134:Catalytic Domain; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology
D001713:Biphenyl Compounds; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D021122:Protein Subunits; D011753:Pyrones; D011994:Recombinant Proteins; D013876:Thiophenes; D000249:Adenosine Monophosphate; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases; C512408:4-hydroxy-3-(4-(2-hydroxyphenyl)phenyl)-6-oxo-7H-thieno(2,3-b)pyridine-5-carbonitrile