title: Purification and characterization of aminopeptidase N from Spodoptera litura expressed in Sf21 insect cells.
abstract: Insecticidal crystal proteins produced by strains of Bacillus thuringiensis cause larval death upon interaction with specific receptors located at the midgut epithelium of susceptible insects. Large quantities of easily purified aminopeptidase and cadherin-like Cry toxin receptors can facilitate the further study of Cry toxin binding and pore formation. Here, we report the solubilisation and purification of aminopeptidase N from Spodoptera litura (SlAPN). Recombinantly expressed and membrane anchored aminopeptidase N showed differential solubilisation with various ionic and nonionic detergents. The N-lauryl sarcosine (NLS)-solubilised SlAPN was purified to near homogeneity by anion exchange and gel filtration chromatography and refolded to its catalytically active form. The optimized purification regimen lead to >90% purification of the catalytically active SlAPN with 11% recovery and 9-folds purification. The interaction of purified SlAPN with biologically active Cry1C protein has been qualitatively and quantitatively characterized. By ligand blotting experiment, we demonstrated the linearity of interaction of the two purified proteins and lack of interaction of SlAPN with structurally divergent nontoxic Cry1Ac protein. The equilibrium dissociation constant (K(D)) of purified SlAPN for Cry1C was calculated by ELISA (90nM). Interaction of enzymatically inactive SlAPN with Cry1C and catalytic activity of APN-Cry1C complex suggested that the catalytic site and toxin-binding sites of SlAPN do not overlap.
authors.affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Ravinder
authors.initials: R
authors.lastname: Kaur
authors.forename: Neema
authors.initials: N
authors.lastname: Agrawal
authors.forename: Raj
authors.initials: R
authors.lastname: Bhatnagar
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism; D018826:CD13 Antigens / Q000096:biosynthesis / Q000302:isolation & purification*; D004731:Endotoxins / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism; D017510:Protein Folding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012995:Solubility; D018411:Spodoptera / Q000201:enzymology
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens