High Level of Soluble Expression in Escherichia coli and Characterisation of the Cloned Bacillus thuringiensis Cry4Ba Domain III Fragment

Chayaratanasin, Poramed; Moonsom, Seangdeun; Sakdee, Somsri; Chaisri, Urai; Katzenmeier, Gerd; Angsuthanasombat, Chanan

doi:10.5483/bmbrep.2007.40.1.058

Cited by 18 publications

(30 citation statements)

References 27 publications

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“…The truncated protein may fail to fold properly or may be unable to form inclusions, and could be completely digested by the proteases of the host. Amino acids at the N-and C-termini of other B. thuringiensis crystal toxins, such as Cry4Ba, also perform a crucial function in inclusion formation and solubilization (15,16). The production of several proteins in E. coli has been improved when expressed as GST-fusion proteins, including mosquitocidal toxin 1 (Mtx1) (17), chitinases (18), and HIV proteins (19).…”

Section: Resultsmentioning

confidence: 99%

Amino acids at N- and C-termini are required for the efficient production and folding of a cytolytic γ-endotoxin from Bacillus thuringiensis

Thammachat

Pathaichindachote²,

Krittanai³

et al. 2008

BMB Reports

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Bacillus thuringiensis Cyt2Aa toxin is a mosquito-larvicidal and cytolytic δ-endotoxin, which is synthesized as a protoxin and forms crystalline inclusions within the cell. These inclusions are solubilized under alkaline conditions and are activated by proteases within the larval gut. In order to assess the functions of the N-and C-terminal regions of the protoxin, several N-and C-terminal truncated forms of Cyt2Aa were constructed. It was determined that amino acid removal at the N-terminal, which disrupts the β1 structure, might critically influence toxin production and inclusion formation. The deletion of 22 amino acids from the C-terminus reduced the production and solubility of the toxin. However, the removal of more than 22 amino acids from the C-terminus or the addition of a bulky group to this region could result in the inability of the protein to adopt the proper folding. These findings directly demonstrated the critical roles of N-and C-terminal amino acids on the production and folding of the B. thuringiensis cytolytic δ-endotoxin. [BMB reports 2008; 41(11): 820-825]

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Section: Resultsmentioning

confidence: 99%

Amino acids at N- and C-termini are required for the efficient production and folding of a cytolytic γ-endotoxin from Bacillus thuringiensis

Thammachat

Pathaichindachote²,

Krittanai³

et al. 2008

BMB Reports

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“…Although an overall topology of this domain is rather similar to some carbohydrate-binding protein domains such as the cellulosebinding domain of a 1,4--glucanase enzyme (Johnson Biochemistry 1999) [28], its functional role is still not clearly elucidated. Nevertheless, it has been implicated in membrane permeabilisation (Masson AEM 2002) [29] or receptor recognition and specificity determination (Burton JMB 1999) [30] (De Maagd AEM 2000) [31] (Chayaratanasin JBMB 2007) [32]. This domain could be also critical for the structural integrity of the toxin molecule as the position of the C-terminus (i.e.…”

Section: Structural Description Of the Three-domain Toxinsmentioning

confidence: 99%

Mosquito Resistance to Bacterial Larvicidal Toxins

Wirth¹

2013

TOTNJ

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Abstract:The insecticidal character of the three-domain Cry -endotoxins produced by Bacillus thuringiensis during sporulation is believed to be caused by their capability to generate lytic pores in the target larval midgut cell membranes. This review describes toxic mechanisms with emphasis on the structural basis of pore formation by two closely related dipteran-specific toxins, Cry4Aa and Cry4Ba, which are highly toxic to mosquito larvae. One proposed toxic mechanism via an "umbrella-like" structure involves membrane penetration and pore formation by the 4-5 transmembrane hairpin. The lipid-induced -conformation of 7 could possibly serve as a lipid anchor required for an efficient insertion of the pore-forming hairpin into the bilayer membrane. Though current electron crystallographic data are still inadequate to provide such critical insights into the structural details of the Cry toxin-induced pore architecture, this pivotal evidence clearly reveals that the 65-kDa active toxin in association with the lipid membrane could exist in at least two different trimeric conformations, implying the closed and open states of a functional pore.

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“…This differential expression could be due to the perturbation of substituted amino acids on structural folding and molecular stability of these mutants. Such a mutation and production of truncated protein, leading to structural alteration, can be found in many other proteins, for example in mutated cytochrome cbb3 oxidase (19) and Cry4Ba toxins (20).…”

Section: Resultsmentioning

confidence: 99%

Amino acid substitution on β and α of Cyt2Aa2 affects molecular interaction of protoxin

Thammachat¹,

Pungtanom²,

Kidsanguan³

et al. 2010

BMB Reports

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Cyt2Aa2 is a mosquito-larvicidal protein produced as a 29 kDa crystalline protoxin from Bacillus thuringiensis subsp. darmstadiensis. To become an active toxin, proteolytic processing is required to remove amino acids from its N-and C-termini. This study aims to investigate the functional role of amino acid residues on the N-terminal β1 and C-terminal αF of Cyt2Aa2 protoxin. Mutant protoxins were constructed, characterized and compared to the wild type Cyt2Aa2. Protein expression data and SDS-PAGE analysis revealed that substitution at leucine-33 (L33) of β1 has a critical effect on dimer formation and structural stability against proteases. In addition, amino acids N230 and I233-F237 around the C-terminus αF demonstrated a crucial role in protecting the protoxin from proteolytic digestion. These results suggested that β1 and αF on the Nand C-terminal ends of Cyt2Aa2 protoxin play an important role in the molecular interaction and in maintaining the structural stability of the protoxin. [BMB reports 2010; 43(6): 427-431]

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High Level of Soluble Expression in Escherichia coli and Characterisation of the Cloned Bacillus thuringiensis Cry4Ba Domain III Fragment

Cited by 18 publications

References 27 publications

Amino acids at N- and C-termini are required for the efficient production and folding of a cytolytic γ-endotoxin from Bacillus thuringiensis

Amino acids at N- and C-termini are required for the efficient production and folding of a cytolytic γ-endotoxin from Bacillus thuringiensis

Mosquito Resistance to Bacterial Larvicidal Toxins

Amino acid substitution on β and α of Cyt2Aa2 affects molecular interaction of protoxin

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