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

摘要: Abstract 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)2SO4 precipitation, filtration Sephadex G-100. The preparation appears to be homogeneous as determined by sedimentation equilibrium studies, isoelectric focusing, amino-terminal amino acid sequence analysis. ability CRP promote selected transcription lost upon elution phosphocellulose but activity usually regained within 20 48 hours. active stable several months stored either frozen or at 4°. weight average molecular native ∼45,000, based data; it composed two apparently identical subunits ∼22,500. constant s20,w = 3.5 was velocity. Isoelectric focusing data show have an point 9.12. Titration free sulfhydryl groups 5,5'-dithiobis(2-nitrobenzoic acid) 4,4'-dithiodipyridine in presence 6 m guanidine-HCl shows four per dimer; these account half-cystines observed