title: Purification of CREB to apparent homogeneity: removal of truncation products and contaminating nucleic acid.
abstract: The cAMP response element binding protein (CREB) is a mammalian transcription factor which regulates the expression of many cellular genes. CREB is commonly expressed in Escherichia coli and purified by heat-extraction followed by affinity chromatography. We have discovered that although this purification yields a reasonably pure product which is active in DNA-binding and functional assays, it contains a large amount of nucleic acid as well as CREB truncation products and other polypeptides. Consequently, this CREB is inadequate for use in biophysical studies including crystallography, and spectroscopic analysis such as analytical ultracentrifugation, FRET, and circular dichroism. We revised the purification protocol to incorporate expression in the Rosetta host strain, nuclease treatment, and denaturing/high salt size-exclusion chromatography. We typically obtain 10mg of CREB per liter of culture media that is 99% homogenous, free of nucleic acid, and amenable to biophysical studies. Comparison of CREB from the original and revised protocols shows similar affinities for the cAMP response element (CRE) but small differences in their secondary structures when assayed by limited proteolysis and circular dichroism.
authors: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.
authors: Dinaida I
authors: DI
authors: Lopez
authors: Jeanne E
authors: JE
authors: Mick
authors: Jennifer K
authors: JK
authors: Nyborg
mesh_terms: D001483:Base Sequence; D002138:Calibration; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers; D008970:Molecular Weight; D009696:Nucleic Acids / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D009696:Nucleic Acids
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