Immobilization of cells with surface-displayed chitin-binding domain

Abstract

To explore chitin-binding domain (ChBD)-based cell immobilization, a tripartite gene fusion consisting of an in-frame fusion of ChBD to lpp and ompA was constructed and expressed in Escherichia coli. ChBD-displayed cells exhibited highly specific and stable binding to chitin within a wide range of pHs (5 to 8) and temperatures (15 to 37 degrees C). These results illustrate the promising use of this approach for engineering applications.

FIG. 1.

(A) Western blot analysis of cellular proteins from strains BL21(DE3)/pLOA and BL21(DE3)/pLOA-ChBD. Plasmid-bearing E. coli strains were cultured and induced by IPTG to initiate the production of the fusion proteins Lpp-OmpA and Lpp-OmpA-ChBD, respectively. At the end of cultivation, cells were harvested by centrifugation and subsequently disrupted by sonication. After centrifugation, the supernatant was removed as the soluble fraction and the insoluble fraction was obtained from the precipitate. A further treatment of insoluble fraction as described previously (17) gave the membrane fraction. Immunoblotting was carried out by first analyzing the fractioned proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis conducted on 12% acrylamide gels as described previously (3). After electrophoresis, protein spots were blotted onto nitrocellulose membranes using a Bio-Rad electrophoretic transfer cell and then subjected to hybridization with a rabbit ChBD-specific antibody (New England Biolabs) at a 1:1,000 dilution for 3 h. After two washes, the horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (Sigma) was administered for 3 h. Finally, another two washes were applied, and the addition of 4-chloro-1-naphthol allowed the HRP-mediated color change to develop. Lane 1, protein marker; lane 2, soluble fraction of the IPTG-induced strain BL21(DE3)/pLOA; lane 3, soluble fraction of strain BL21(DE3)/pLOA-ChBD; lane 4, soluble fraction of the IPTG-induced strain BL21(DE3)/pLOA-ChBD; lane 5, insoluble fraction of the IPTG-induced strain BL21(DE3)/pLOA; lane 6, insoluble fraction of strain BL21(DE3)/pLOA-ChBD; lane 7, insoluble fraction of the IPTG-induced strain BL21(DE3)/pLOA-ChBD; lane 8, membrane fraction of the IPTG-induced strain BL21(DE3)/pOLA; lane 9, membrane fraction of strain BL21(DE3)/pLOA-ChBD; lane 10, membrane fraction of the IPTG-induced strain BL21(DE3)/pLOA-ChBD. The arrow indicates the position of Lpp-OmpA-ChBD. (B) Colony blotting of strains BL21(DE3)/pLOA and BL21(DE3)/pLOA-ChBD. As modified from the previous report (7), one drop of cell equilibrant (OD₅₅₀ of 10) was layered on nitrocellulose membranes. Subsequent incubation with anti-ChBD antibodies was conducted for 1 h and followed by a gentle wash. After that, the color development mediated by HRP was carried out by mixing the anti-rabbit IgG and 4-chloro-1-naphthol. Panel 1, IPTG-induced strain BL21(DE3)/pLOA; panel 2, strain BL21(DE3)/pLOA-ChBD; panel 3, IPTG-induced strain BL21(DE3)/pLOA-ChBD.

FIG. 2.

Electron micrographs of chitin beads used for cell immobilization. Refer to the text for details. Strains BL21(DE3)/pLOA (control) and BL21(DE3)/pLOA-ChBD were cultured and induced for hybrid protein production. Subsequently, the induced cells and the induced strain BL21(DE3)/pLOA-ChBD, treated with trypsin, were prepared for the adsorption experiment, and the result was visualized by VVSEM. The trypsin-treated strain BL21(DE3)/pLOA-ChBD gave a cell binding result similar to that of the control strain (data not shown). (A) IPTG-induced strain BL21(DE3)/pLOA-ChBD. (B) IPTG-induced strain BL21(DE3)/pLOA.

FIG. 3.

(A) Dependence of cell immobilization on pH. The binding of the IPTG-induced strain BL21(DE3)/pLOA-ChBD to chitin was carried out at 4°C for 24 h in 20 mM buffer solution at the pHs indicated. After the termination of adsorption, the removed chitin received an extensive wash, and the number of cells remaining in the solution was then determined. The amount of WCW or OD thus measured relative to that before adsorption was expressed as the percentage of cells adsorbed. The adsorption solution used here included sodium citrate buffer (pH 5 and 6), sodium phosphate buffer (pH 7), Tris-HCl buffer (pH 8), and glycine-sodium hydroxide buffer (pH 9 and 10). (B) Effect of buffer concentration on cell binding. In a similar fashion, cells were adsorbed to chitin in sodium phosphate buffer (pH 7.0) at various concentrations. For parallel comparison, the pH of double-distilled water (ddH₂O) was adjusted to 7.0. The data shown represent three independent experiments.

FIG. 4.

(A) Dependence of cell immobilization on temperature. Similar to the adsorption conditions described in the legend to Fig. 3, cell binding was performed in 50 mM sodium phosphate buffer (pH 7.0) at various temperatures for 24 h. (B) Effect of adsorption time on cell binding. The adsorption conditions remained unchanged, although the temperature was kept at 4°C for various times. The data were adopted from three independent experiments.