Overproducing araC protein with lambda-arabinose transducing phage.

Abstract

Escherichia coli infected with bacteriophage lambda-arabinose transducing phage were tested as sources of araC protein. Infection of cells with such phage produces an intracellular concentration of araC protein up to 100 times that present in wild-type E. coli, apparently resulting from fusion of the araC gene to bacteriophage lambda promoters. Lysates from these phage-infected cells may be fractionated to yield another 100-fold enrichment in araC activity so that the total enrichment is 10,000-fold. A nonsense mutation in araC provided proof of the identification on gel electrophoresis of a band in the purified material. Biologically active araC protein is a dimer with 28,000 M.W. subunits. The araC gene in these phage replaces the int-xis genes but is oriented in the opposite direction. Nonetheless, it appears to be transcribed in this position by the phage promoter pr via transcription the long way around. Furthermore, because araC gene is in this position, we were able to isolate phage on which the araC gene was under phage late gene control by deletion of the late gene transcription stop signals in the b2 region.