title: 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.
authors.affiliation: Laboratory of Structural Biochemistry, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phuthamontol 4 Road, Nakornpathom 73170, Thailand.
authors.forename: Anon
authors.initials: A
authors.lastname: Thammasittirong
authors.forename: Manasave
authors.initials: M
authors.lastname: Dechklar
authors.forename: Somphob
authors.initials: S
authors.lastname: Leetachewa
authors.forename: Kusol
authors.initials: K
authors.lastname: Pootanakit
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D000330:Aedes / Q000201:enzymology* / Q000235:genetics; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000201:enzymology; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D058851:GPI-Linked Proteins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000469:Alkaline Phosphatase