RESUMO
We have developed a protocol to produce large quantities of high purity myristoylated and non-myristoylated neuronal calcium sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium-dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6x His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100-200 microM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50mg of homogenous myristoylated NCS-1 is obtained from 1L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 DeltaEF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.
Assuntos
Aciltransferases/metabolismo , Escherichia coli/metabolismo , Ácido Mirístico/metabolismo , Proteínas Sensoras de Cálcio Neuronal/biossíntese , Proteínas Sensoras de Cálcio Neuronal/isolamento & purificação , Neuropeptídeos/biossíntese , Neuropeptídeos/isolamento & purificação , Aciltransferases/genética , Sítios de Ligação , Cálcio/química , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/química , Escherichia coli/genética , Técnicas de Transferência de Genes , Humanos , Lipídeos/química , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Ácido Mirístico/química , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Isoformas de Proteínas/química , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de Tempo , Leveduras/enzimologiaRESUMO
Frequenin is a member of the neuronal Ca(2+) sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca(2+)-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca(2+) myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca(2+) myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca(2+)-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca(2+)-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.
