Isolation of bacterial and phage proteins by homopolymer RNA-cellulose chromatography.
Nucleic acid-free extracts of Escherichia coli have been analyzed by chromatography on columns of cellulose, to which poly(A), poly(U), or poly(C) have been attached by ultraviolet irradiation. Proteins are released from the columns by stepwise elution with increasingly higher concentrations of salt, followed by washing with urea to remove very tightly bound molecules. The pattern of protein elution is reproducibly different for each of the homopolymer RNA-cellulose columns used: some proteins bind very tightly to one column, but poorly to others. Analysis by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, by immunological cross-reactivity in double diffusion tests, and by enzymological assays, has allowed the identification of a number of these proteins. The RNA polymerase core enzyme binds to poly(C)- and to poly(U)-cellulose columns, and can be purified to 20 to 30 percent homogeneity in a single step. Ribosomal protein S1 and the termination factor rho bind very tightly to poly(C)-cellulose, and both can be purified to homogeneity rapidly, in much higher yields than previously reported. Poly(A)-cellulose chromatography allows the isolation of large amounts of an 80,000 molecular weight protein having an as yet unassigned cellular function. The host factor required for RNA phage Qbeta RNA replication in vitro can also be obtained from poly(A)-cellulose, and chromatography of extracts of phage Qbeta-infected E. coli on RNA-cellulose columns results in very rapid isolation of the Qbeta replicase enzyme. Homopolymer RNA-cellulose chromatography thus appears to be a simple, general technique, useful for the efficient isolation of a variety of RNA-binding proteins.
D001426:Bacterial Proteins / Q000302:isolation & purification*; D001665:Binding Sites; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003090:Coliphages / Q000032:analysis / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis; D005779:Immunodiffusion; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification; D011485:Protein Binding; D011777:Q beta Replicase / Q000276:immunology / Q000302:isolation & purification*; D012313:RNA; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D012328:RNA Viruses / Q000032:analysis / Q000201:enzymology; D014764:Viral Proteins / Q000302:isolation & purification*
D001426:Bacterial Proteins; D010448:Peptide Initiation Factors; D014764:Viral Proteins; D012313:RNA; D011777:Q beta Replicase; D012316:RNA Nucleotidyltransferases