Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis

Srisucharitpanit, Kanokporn; Inchana, Pattarapong; Rungrod, Amporn; Promdonkoy, Boonhiang; Boonserm, Panadda

doi:10.1016/j.pep.2012.02.009

Cited by 17 publications

(9 citation statements)

References 29 publications

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“…Cry49Aa1 clearly shows significant similarity to Cry35Aa1, particularly in the core, β-sheet region of the C-terminal domain (Figure 7B). The high proportion of β-sheet structure in Cry35Ab1 is consistent with CD analysis of the related BinA protein that indicated a high proportion of β-sheet and little α-helix [48], [49], [50].…”

Section: Resultssupporting

confidence: 78%

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Kelker¹,

Berry

Evans³

et al. 2014

PLoS ONE

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Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.

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Section: Resultssupporting

confidence: 78%

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Kelker¹,

Berry

Evans³

et al. 2014

PLoS ONE

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“…However, the precise nature of the oligomers, if any, formed by the subunits before and after activation is still controversial. Earlier studies show striking discrepancies, e.g., formation of heterotetramers (BinA 2 -BinB 2 ) for protoxins and no interaction of their activated subunits [ 22 ], complete formation of 1:1 complexes between activated subunits at low μM concentration [ 23 ], or weak interaction between activated subunits, with k a ~5 × 10 3 M −1 measured by surface plasmon resonance (SPR), i.e., a 5 mM activated toxin would produce 50% heterodimer [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation

et al. 2016

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The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

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“…As reported previously, the expression of recombinant BinB protein as a soluble form in E. coli was achieved by fusing the protein with a hexahistidine tag at the N-terminus and modifying the strategy of cell culture by reducing temperature (Srisucharitpanit et al, 2012). The in vitro activation of the recombinant BinB was performed by trypsin digestion to generate a resistant fragment of 45 kDa which is fully active against C. quinquefasciatus larvae.…”

Section: Resultsmentioning

confidence: 99%

“…Expression, purification and activation of native and selenomethionine-substituted BinB Detailed cloning, expression and purification protocols for the trypsin-activated BinB have been described previously (Srisucharitpanit et al, 2012). Briefly, the binB gene from B. sphaericus strain 2297 (GenBank accession No.…”

Section: Methodsmentioning

confidence: 99%

Crystallization and preliminary X-ray crystallographic analysis of the functional form of BinB binary toxin fromBacillus sphaericus

et al. 2013

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The binary toxin from Bacillus sphaericus consists of two proteins, BinA and BinB, which work together to exert toxicity against mosquito larvae. BinB is proposed to be a receptor-binding domain and internalizes BinA into the midgut cells, resulting in toxicity via an unknown mechanism. The functional form of BinB has been successfully crystallized. The crystals of BinB diffracted to a resolution of 1.75 Å and belong to space group P6 2 22, with unit-cell parameters a = b = 95.2, c = 154.9 Å . Selenomethionine-substituted BinB (SeMetBinB) was prepared and crystallized for experimental phasing. The SeMetBinB crystal data were collected at a wavelength of 0.979 Å and diffracted to a resolution of 1.85 Å .

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Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis

Cited by 17 publications

References 29 publications

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation

Crystallization and preliminary X-ray crystallographic analysis of the functional form of BinB binary toxin fromBacillus sphaericus

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