title: Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis.
abstract: The binary toxin produced from Bacillus sphaericus is highly toxic against larvae of Culex and Anopheles mosquitoes. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB, which are produced as crystalline inclusions during sporulation. Currently, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. Herein, we describe an expression protocol with modified conditions allowing production of soluble, biologically active BinA and BinB for further structural analysis. The binA and binB genes from B. sphaericus 2297 strain were independently cloned and fused with a polyhistidine tag at their N-termini. Both (His)(6)-tagged BinA and (His)(6)-tagged BinB were expressed as soluble forms at low temperature. Highly pure proteins were obtained after two-step purification by Ni-NTA affinity and size exclusion chromatography. In vitro activation by trypsin digestion generated a resistant fragment, of 40kDa for BinA, and of 45kDa for BinB, and an oligomeric complex of BinA and BinB in solution was observed after proteolytic activation. Their functional and structural properties were confirmed by a biological assay and far-UV circular dichroism, respectively. The mixture of BinA and BinB, either as a protoxin or as a trypsin-activated form, exhibited high mosquito-larvicidal activity against Culex quinquefasciatus larvae with LC(50) of about 10ng/ml, while no toxicity was observed from the single binary toxin component. Results from far-UV circular dichroism of BinA and BinB suggest the presence of mainly β-structure. The expression and purification protocols reported here will be useful for the production of the active and homogeneous binary toxin to allow further detailed structural investigation.
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand.
authors.forename: Kanokporn
authors.initials: K
authors.lastname: Srisucharitpanit
authors.forename: Pattarapong
authors.initials: P
authors.lastname: Inchana
authors.forename: Amporn
authors.initials: A
authors.lastname: Rungrod
authors.forename: Boonhiang
authors.initials: B
authors.lastname: Promdonkoy
authors.forename: Panadda
authors.initials: P
authors.lastname: Boonserm
mesh_terms: D000818:Animals; D001407:Bacillus; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D002850:Chromatography, Gel; D003465:Culex / Q000187:drug effects; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D007814:Larva / Q000187:drug effects; D007928:Lethal Dose 50; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007306:Insecticides; D011993:Recombinant Fusion Proteins; C046134:binB protein, Bacillus sphaericus