Purification of and Properties of the Cyclic Adenosine 3’,5’- Monophosphate Receptor Protein which Mediates Cyclic Adenosine 3’, S-Monophosphate-dependent Gene Transcription in Escherichia coli

A procedure is described for the purification of cyclic adenosine 3’,5’-monophosphate receptor protein (CRP) from Escherichia coli involving chromatography on DEAE-cellulose and phosphocellulose, (NH4)&04 precipitation, and filtration on Sephadex G-100. The preparation appears to be homogeneous as determined by sedimentation equilibrium studies, isoelectric focusing, and amino-terminal amino acid sequence analysis. The ability of CRP to promote selected transcription is lost upon elution of the protein from phosphocellulose but activity is usually regained within 20 to 48 hours. The active CRP preparation is stable for several months stored either frozen or at 4”. The weight average molecular weight of the native protein is -45,000, based on sedimentation equilibrium data; it is composed of two apparently identical subunits of molecular weight -22,500. A sedimentation constant ~20,~ = 3.5 was determined by sedimentation velocity. Isoelectric focusing data show the protein to have an isoelectric point of 9.12. Titration of free sulfhydryl groups by either 5,5’-dithiobis(2-nitrobenzoic acid) or 4,4’-dithiodipyridine in the presence of 6 M guanidine-HCl shows the presence of four free sulfhydryl groups per native dimer; these account for the four half-cystines observed by amino acid analysis.