Overexpression and purification of transcriptionally competent CREB from a recombinant baculovirus.
Signal transduction and viral stimulatory pathways converge ultimately at the level of transcriptional activation to influence the expression of a variety of cellular genes in response to environmental stimuli and developmental signals. Recent studies have implicated the cyclic AMP-responsive element-binding protein (CREB) to be involved in mediating transcriptional activation in response to multiple varied stimuli, including (1) stimulation of the protein kinase A signal transduction pathway; (2) membrane depolarization and increases in intracellular calcium; and (3) viral induced gene expression. In order to study the structure and functional mechanisms of CREB actions in these systems, full-length CREB-327 was expressed in Spodoptera frugiperda (Sf9) cells with the baculovirus expression vector system. The expressed CREB, which is phosphorylated and localized in the nucleus, is capable of enhancing the transcription of a reporter gene containing the CRE sequence in a cell-free transcription assay. Approximately 12.5 mg of purified CREB per liter of infected Sf9 cell culture can be obtained. These large amounts of purified protein will facilitate studies of the structure and functions of this important transcriptional regulatory protein.
Chen
C Y
CY
Department of Medicine, University of Colorado School of Medicine, Denver 80262.
Bessesen
D H
DH
Jackson
S M
SM
Hoeffler
J P
JP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D005591:Chemical Fractionation; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D006358:Hot Temperature; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors