title: Single-column purification of the tag-free, recombinant form of the neuronal calcium sensor protein, hippocalcin expressed in Escherichia coli.
abstract: Hippocalcin is a 193 aa protein that is a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. Mice that lack the function of this protein are compromised in the long term potentiation aspect of memory generation. Recently, mutations in the gene have been linked with dystonia in human. The protein has no intrinsic enzyme activity but is known to bind to variety of target proteins. Very little information is available on how the protein executes its critical role in signaling pathways, except that it is regulated by binding of calcium. Further delineation of its function requires large amounts of pure protein. In this report, we present a single-step purification procedure that yields high quantities of the bacterially expressed, recombinant protein. The procedure may be adapted to purify the protein from inclusion bodies or cytosol in its myristoylated or non-myristoylated forms. MALDI-MS (in source decay) analyses demonstrates that the myristoylation occurs at the glycine residue. The protein is also biologically active as measured through tryptophan fluorescence, mobility shift and guanylate cyclase activity assays. Thus, further analyses of hippocalcin, both structural and functional, need no longer be limited by protein availability.
authors.affiliation: Graduate School of Biomedical Sciences, Rowan University, Stratford, NJ 08084, USA.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Krishnan
authors.affiliation: Graduate School of Biomedical Sciences, Rowan University, Stratford, NJ 08084, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Viviano
authors.affiliation: Graduate School of Biomedical Sciences, Rowan University, Stratford, NJ 08084, USA.
authors.forename: Yaroslav
authors.initials: Y
authors.lastname: Morozov
authors.affiliation: Graduate School of Biomedical Sciences, Rowan University, Stratford, NJ 08084, USA; School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084, USA. Electronic address: vvenkat2007@gmail.com.
authors.forename: Venkat
authors.initials: V
authors.lastname: Venkataraman
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D051597:Hippocalcin / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C496693:Hpca protein, rat; D011994:Recombinant Proteins; D051597:Hippocalcin
keywords: Calcium; EF hands; Hippocalcin; Memory; Myristoylation; Neuronal calcium sensor protein