title: Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, P.O. Box 850, Hershey, PA 17033, USA.
authors.forename: Dan A
authors.initials: DA
authors.lastname: De Cotiis
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Woll
authors.forename: Todd E
authors.initials: TE
authors.lastname: Fox
authors.forename: R Blake
authors.initials: RB
authors.lastname: Hill
authors.forename: Robert
authors.initials: R
authors.lastname: Levenson
authors.forename: John M
authors.initials: JM
authors.lastname: Flanagan
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D008055:Lipids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism*; D051580:Neuronal Calcium-Sensor Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D009479:Neuropeptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D020033:Protein Isoforms / Q000737:chemistry; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors; D015003:Yeasts / Q000201:enzymology
chemical_list: D008055:Lipids; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D020033:Protein Isoforms; C101464:frequenin calcium sensor proteins; D019814:Myristic Acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D002118:Calcium