Purification of CREB to apparent homogeneity: removal of truncation products and contaminating nucleic acid.
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.
Lopez
Dinaida I
DI
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.
Mick
Jeanne E
JE
Nyborg
Jennifer K
JK
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
D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D009696:Nucleic Acids
J Mol Biol. 1999 Oct 22;293(2):321-31
10550212
J Biol Chem. 2007 Jul 6;282(27):19872-83
17491014
Proc Natl Acad Sci U S A. 1990 Jul;87(14):5258-62
2142528
Metabolism. 1990 Sep;39(9 Suppl 2):13-6
2144888
Mol Cell Biol. 1991 Mar;11(3):1306-12
1671708
Mol Endocrinol. 1991 Feb;5(2):256-66
1828107
EMBO J. 1992 Apr;11(4):1503-12
1532935
Proc Natl Acad Sci U S A. 1993 May 15;90(10):4606-10
8506306
Protein Sci. 1993 Sep;2(9):1461-71
8401230
J Biol Chem. 1993 Oct 5;268(28):21225-31
8407959
Nature. 1993 Oct 28;365(6449):855-9
8413673
Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9046-50
8105470
Oncogene. 1994 Feb;9(2):463-8
8290258
Nature. 1994 Jul 21;370(6486):223-6
7913207
Nature. 1994 Jul 21;370(6486):226-9
8028671
Nature. 1995 Mar 2;374(6517):81-4
7870178
Nature. 1995 Mar 2;374(6517):85-8
7870179
J Biol Chem. 1996 Jun 7;271(23):13716-23
8662719
Cell Signal. 2004 Nov;16(11):1211-27
15337521