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Title: Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis
Authors: Kanokporn Srisucharitpanit
Pattarapong Inchana
Amporn Rungrod
Boonhiang Promdonkoy
Panadda Boonserm
Mahidol University
Thailand National Center for Genetic Engineering and Biotechnology
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Apr-2012
Citation: Protein Expression and Purification. Vol.82, No.2 (2012), 368-372
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 40 kDa for BinA, and of 45 kDa 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 10 ng/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. © 2012 Elsevier Inc. All rights reserved.
ISSN: 10465928
Appears in Collections:Scopus 2011-2015

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