FIG. 2.

Effect of periplasmic 1C11 scFv on colicin A activity. (A) Subcellular location of the 1C11 scFv produced in E. coli. Cells were grown at 37°C to an A₆₀₀ of 0.5 and then induced with the indicated concentration of IPTG for 20 min. The amount of scFv in the periplasmic fraction (p) and spheroplast fraction (sph) was estimated by immunoblotting with MAb 9E10 (directed against the c-myc tag). (B) 1C11 scFv binding in the periplasmic fraction in vitro. An ELISA was used to measure the binding activity of the 1C11 scFv in the periplasmic fraction. Colicin A or bovine serum albumin was used to coat the plates at a final concentration of 100 ng of protein/well, and then the plates were treated with periplasmic fractions. HRP-labelled rabbit anti-mouse IgG was used as the second antibody. Bound antibodies were detected by the optical density (OD) at 405 nm. (C) In vivo inhibition of colicin A activity by 1C11 scFv produced in sensitive cells. 1C11 scFv-producing cells and control cells were prepared and induced as described above and then were suspended in 100 mM sodium phosphate buffer (pH 7.2). Colicin A was added at a multiplicity (number of colicin molecules per cell) of 100 (shaded bars) or 10⁵ (hatched bars), and the initial rate of K⁺ efflux was determined from the linear part of the K⁺ efflux curve. The relative inhibition of K⁺ efflux was calculated as described in Materials and Methods. Mean values and standard errors are shown; three independent experiments were performed for each set of conditions.