IMMUNOCHEMICAL DETERMINATION OF THE CELLULAR CONTENT OF POLYPEPTIDE-CHAIN RELEASE FACTOR RF3 IN ESCHERICHIA-COLI

Polypeptide chain termination in Escherichia coli is known to require two codon specific release factors, RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigation s have estimated the cellular content of factors RF1 and RF2. Two diff erent immunological techniques for measuring the amount of RF3 per cel l in crude E coli cell extracts are reported here, using a sensitive i mmunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extens ively purified recombinant E coli RF3. The immunoblotting involves a s pecific monoclonal antibody (mAb), biotinylated second antibody and fi nally radioactive iodinated streptavidin. In the sandwich assay polycl onal antibodies are immobilised on a polystyrene surface before additi on of crude cell extract; a specific mAb serves as primary antibody an d an HRP-labelled anti-mouse Ig as secondary antibody. Both methods ar e accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably a ccording to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively c orrelated with the number of RF3 molecules per cell.