Abstract
Regulation of AMP-activated protein kinase (AMPK) signalling is complex and involves contributions from adenine nucleotides, co-/posttranslational modifications, and isoform composition of the AMPK heterotrimer. It is becoming apparent that AMPK activation/inhibition by synthetic drugs involves similar levels of complexity. Major advances in our understanding of these mechanisms have been gained from recombinant expression systems that provide sufficient quantities of highly purified material for structure/function studies. Here, we provide a detailed protocol for transient expression of affinity-tagged AMPK complexes in mammalian cells. We have found this system to be optimal as a source of enzyme possessing regulatory modifications found in vivo.
Keywords:
Biochemistry; Kinase; Mammalian cell; Metabolism; Purification; Recombinant protein; Transfection.
MeSH terms
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AMP-Activated Protein Kinases / chemistry
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AMP-Activated Protein Kinases / isolation & purification
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AMP-Activated Protein Kinases / metabolism*
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Adenine Nucleotides / metabolism*
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Affinity Labels / chemistry
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Animals
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COS Cells
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Chlorocebus aethiops
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Enzyme Activation
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Enzyme Assays / instrumentation
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Enzyme Assays / methods*
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HEK293 Cells
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Humans
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Isoenzymes / chemistry
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Isoenzymes / isolation & purification
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Isoenzymes / metabolism
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Phosphorylation
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Protein Processing, Post-Translational
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Protein Subunits / chemistry
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Protein Subunits / isolation & purification
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Protein Subunits / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Signal Transduction
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Staining and Labeling / methods
Substances
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Adenine Nucleotides
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Affinity Labels
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Isoenzymes
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Protein Subunits
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Recombinant Proteins
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AMP-Activated Protein Kinases