A protocol for expression and purification of cyclic nucleotide-free protein in Escherichia coli

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are ubiquitous second messengers that relay signals in almost all forms of life. Binding of cyclic nucleotides to their receptors results in subsequent modulation of the receptor’s activity, which propagates the signal to downstream effectors. Although the rich structural diversity occurring among these receptors includes drastically different folds such as G-Protein-Couped-Receptors (GPCRs) and cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA (GAF) domains, the canonical cyclic nucleotide-binding (CNB) domain is the most common and well characterized. CNB domains are ancient domains occurring in diverse multidomain proteins such as kinases, transcription factors, ion channels, nucleotidyl cyclases, guanine exchange factors, and acetyltransferases, often functioning as allosteric modulators of enzyme activity. These domains serve as conformational switches that structurally rearrange upon binding of the cyclic nucleotides. In mammalian physiology, cyclic nucleotides are critical second messengers that relay signals through these enzymes, regulating diverse physiological processes such as vision, learning and memory, vasodilation, and metabolism. Consistent with their importance in physiology, mutations in proteins that relay cyclic nucleotide signaling have been linked to a diverse cohort of diseases including achromatopsia, epilepsy, Carney complex, fibrolamellar hepatocellular carcinoma, thoracic aortic aneurysms, and acute aortic dissections. The universal importance of cyclic nucleotide signaling in the normal function of mammalian cells has stimulated decades of research into the CNB domain-containing proteins that relay these signals and firmly established some of these proteins as drug targets in the treatment of disease.