Article Figures & Data
Figures
- Fig. 1.
Construction of the pscFv1C11 expression vector. (A) Position of the 1C11 epitope in the translocation domain of colicin A. T, RB, and PF, translocation, receptor-binding, and pore-forming domains of colicin A, respectively. The amino acid residues are abbreviated as follows: E, Glu; G, Gly; P, Pro; R, Arg; S, Ser; T, Thr; and W, Trp. (B) pscFv1C11 expression vector. The gene coding for the 1C11 scFv (VH, L, VL) was amplified by PCR and inserted into pPelB35PhoA′. The bla,lacIq, SS, VH, L, VL, and c-myc genes encode the β-lactamase, lacrepressor, signal sequence of pectate lyase of Erwinia carotovora, VH domain, linker [(Gly4Ser)3], VL domain, and c-myc tag, respectively. The scFv was under the control of the IPTG-inducible tac promoter (Ptac).
- 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 A600of 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.
- Fig. 3.
Effect of DTT on periplasmic 1C11 scFv. (A) Amount of 1C11 scFv in periplasmic extracts. Periplasmic extracts were prepared as described in the legend to Fig. 2A and Materials and Methods, except that cells were incubated with the indicated concentration of DTT during induction (20 μM IPTG). (B) In vitro binding of periplasmic 1C11 scFv. Periplasmic extracts containing the 1C11 scFv were tested by ELISA. Bars indicate the optical density (OD) at 405 nm. (C) In vivo binding of the 1C11 scFv. The inhibition of colicin A activity was determined as described in the legend to Fig. 2C and Materials and Methods, except that uninduced cells were incubated with the indicated concentration of DTT for 20 min before harvesting. Bars indicate the percentage of inhibition. Mean values and standard errors are shown; three independent experiments were performed for each condition.
- Fig. 4.
1C11 scFv specificity. 1C11 and 5A4 scFv-producing cells were prepared as described in the legend to Fig. 3C, except that cells were not incubated with DTT. Inhibition of colicin A, E1, B, AAB, and BBA activity in 1C11 scFv-producing cells and inhibition of colicin A activity in 5A4 scFv-producing cells were determined as described in the legend to Fig. 2C (shaded bars). Cells producing the 1C11 scFv were also incubated with colicin A at a multiplicity of 400. After 1 min, colicin E1 was added at the same multiplicity, and the K+efflux was determined as described above (hatched bar). Bars indicate the percentage of inhibition. Mean values and standard errors are shown; three independent experiments were performed for each condition.
