Aedes aegypti membrane-bound alkaline phosphatase expressed in Escherichia coli retains high-affinity binding for Bacillus thuringiensis Cry4Ba toxin

Abstract

Glycosylphosphatidylinositol-linked alkaline phosphatase (GPI-ALP) from the epithelial membrane of the larval midgut of Aedes aegypti was previously identified as a functional receptor of the Bacillus thuringiensis Cry4Ba toxin. Here, heterologous expression in Escherichia coli of the cloned ALP, lacking the secretion signal and GPI attachment sequences, and assessment of its binding characteristics were further investigated. The 54-kDa His tag-fused ALP overexpressed as an inclusion body was soluble when phosphate buffer (pH 7.5) was supplemented with 8 M urea. After renaturation in a nickel-nitrilotriacetic acid (Ni-NTA) affinity column, the refolded ALP protein was able to retain its phosphatase activity. This refolded ALP also showed binding to the 65-kDa activated Cry4Ba toxin under nondenaturing (dot blot) conditions. Quantitative binding analysis using a quartz crystal microbalance revealed that the purified ALP immobilized on a gold electrode was bound by the Cry4Ba toxin in a stoichiometry of approximately 1:2 and with high affinity (dissociation constant [K(d)] of ∼14 nM) which is comparable to that calculated from kinetic parameters (dissociation rate constant [k(off)]/binding constant [k(on)]). Altogether, the data presented here of the E. coli-expressed ALP from A. aegypti retaining high-affinity toxin binding support our notion that glycosylation of this receptor is not required for binding to its counterpart toxin, Cry4Ba.

Fig. 1.

Detection and characterization of His tag-fused Aa-mALP expressed in E. coli. (A) SDS-PAGE analysis (Coomassie brilliant blue-stained 10% gel) of the expressed Aa-mALP protein. Lane M, broad-range protein markers; lane 1, protein expression pattern of IPTG-induced E. coli cells (∼10⁷ cells) containing pET-Aa-mALP; lane 2, the His tag-fused Aa-mALP purified by elution through a HisTrap column. (B) Western blot analysis of His tag-fused Aa-mALP; lane M, prestained protein standard; lane 1, the purified His tag-fused Aa-mALP protein detected with anti-His (C-terminal) antibody. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gels in panels A and B. (C) Phosphatase activity assay via BCIP/NBT qualitative dot blotting of 1, 3, and 5 μg of the purified His tag-fused Aa-mALP.

Fig. 2.

Dot blot-based analysis of binding between Aa-mALP and the Cry4Ba toxin. The purified refolded Aa-mALP (0.1 or 1 μg) and calf intestinal alkaline phosphatase (CIP) (1 μg) were spotted in duplicate on nitrocellulose membranes which were then incubated with the activated Cry4Ba toxin (1.5 nM). The binding signals were detected as described in Materials and Methods.

Fig. 3.

(A) Normalized changes in resonant frequency upon addition of the purified activated Cry4Ba toxin onto the QCM sensor cell precoated with the His tag-fused Aa-mALP. (Inset) Cry4Ba toxin after trypsin activation showing 47- and 18-kDa trypsin-resistant fragments (lane 1). The gel is as described in the legend to Fig. 1. (B) Linear regression plot of the concentration of Cry4Ba and the concentration of Cry4Ba divided by ΔF. The x-axis intercept is −K_d. (C) Linear regression plot of the concentration of Cry4Ba and 1/relaxation time (τ⁻¹). The y-axis intercept is k_off, and the slope of the line is k_on. Values in panels B and C are the means ± standard errors of the means (SEM; error bars) for three replicate tests.