| Description |
A functional Escherichia coli groNB (nusB) gene has been previously shown to be necessary for bacteriophage lambda N protein function in vivo. The product of the groNB gene has been identified as a polypeptide of approximately 14,000 daltons with an isoelectric point of about 7.0. This was accomplished after infection of UV-irradiated bacteria with various (lamda)groNB+ transducing phage and their groNB- derivatives. Transducing phage carrying either a deletion or an amber mutation in the groNB gene fail to synthesize the 14,000 dalton protein after infection of a sup+ host. The bacterial DNA region containing the groNB gene has been subcloned as 7,700, 4,300, 1,400, or 580 base pair fragments into various plasmids. In each case the recombinant plasmid was able to transform groNB- bacteria to Gro+ demonstrating that the plasmids synthesize a functional groNB gene product. Furthermore, we were able to demonstrate that each recombinant plasmid coded for the in vitro synthesis of the 14,000 dalton groNB protein. We interpret these results to mean that the GroNB- phenotype of bacterial hosts, such as M72groNB115, is due to an inability to produce a functional 14,000 dalton groNB protein. Using a host which harbors the overproducer plasmid, pMOBgroNB a simple three step purification of the groNB protein has been achieved. The purification scheme consists of a high speed centrifugation step followed by ammonium sulfate precipitation and G50 Sephadex column chromatography. The protein obtained after these steps has been shown to be at least 98% pure with no single contaminant comprising more than 2% of the total protein. An in vitro assay, which monitors the N protein mediated antitermination function of lambda and is dependent on exogenous added groNB protein, has been devised. Using this assay system it has been shown that the purified groNB protein is partially active. The purified groNB protein was used to immunize rabbits and to raise antibodies against it. Such antibodies have been shown to precipitate the groNB protein from gro+ but not from groNB- cellular extracts. |
