Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli.
The 5'-AMP-activated protein kinase (AMPK) plays a critical role in the regulation of cellular energy homeostasis. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). To date, purified AMPK has only been obtained in small, microgram quantities from tissues. Here, we describe an expression and purification system for production of functional AMPK in Escherichia coli. A plasmid carrying all three subunits of AMPK (alpha1, beta1, and gamma1) for T7 RNA polymerase-driven transcription of a single tricistronic messenger was constructed, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. AMPK was purified from the bacterial lysates by single-step nickel-ion chromatography, utilizing a poly-histidine tag fused to the N-terminus of the alpha-subunit. The recombinant AMPK complex was monodisperse, as shown by gel filtration chromatography with elution of a single peak at a Stokes radius of 52A. Bacterially expressed AMPK was entirely inactive, yet it could be activated by upstream kinase in the presence of AMP. Sufficient quantities of purified functional AMPK should prove to be an invaluable tool to solve many of the pertinent questions about its molecular structure and function, in particular facilitating protein crystallization for X-ray structure analysis.
Neumann
Dietbert
D
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
Woods
Angela
A
Carling
David
D
Wallimann
Theo
T
Schlattner
Uwe
U
D000249:Adenosine Monophosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008322:Mammals; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D021122:Protein Subunits; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D000263:Adenylate Kinase