Cloning and Characterization of a Cytolytic and Mosquito Larvicidal δ-Endotoxin from Bacillus thuringiensis subsp. darmstadiensis

Promdonkoy, Boonhiang; Chewawiwat, Namchai; Tanapongpipat, Sutipa; Luxananil, Plearnpis; Panyim, Sakol

doi:10.1007/s00284-002-3823-5

Cited by 32 publications

(36 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The gene was placed downstream of the lac promoter in pGEM-Teasy vector and transformed into E. coli JM109, so that it could be induced by IPTG. The full-length Cyt2Aa2 was abundantly generated as inclusion bodies within the E. coli cells (4). These inclusions can be readily solubilized in alkaline buffers such as 50 mM Na2CO3 pH 10.5.…”

Section: Resultsmentioning

confidence: 99%

“…darmstadiensis has been cloned and sequenced in our laboratory (4). It comprises 259 amino acids, identical to that of Cyt2Aa1 (14).…”

Section: Resultsmentioning

confidence: 99%

“…The toxins in the other group, the Cyt toxins, are specifically toxic to Dipteran larvae including mosquito and blackfly larvae, in vivo. The toxins in this group also evidence cytolytic activity to a broad range of cells, including erythrocytes, in vitro (3)(4)(5)(6). The amino acid sequences of all Cyt toxins show a high degree of homology with each other (7), and therefore all of them should evidence similar 3D structures.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

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]

show abstract

Section: Resultsmentioning

confidence: 99%

“…darmstadiensis has been cloned and sequenced in our laboratory (4). It comprises 259 amino acids, identical to that of Cyt2Aa1 (14).…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…The structural folding is composed of six α-helices and seven β-sheets (12). Even though Cyt2 is produced as a crystal protein in Bacillus thuringiensis, it can be cloned and expressed in E. coli as a protein inclusion (10). These crystal and inclusion proteins are solubilized in an alkaline condition of the larval midgut.…”

Section: Introductionmentioning

confidence: 99%

“…Based on amino acid sequence similarity, the toxins can be divided into two classes: Cyt1 and Cyt2. They share 39% and 70% amino acid sequence identity and similarity, respectively (8)(9)(10)(11). Expression of Cyt1 toxin requires P20 helper protein, while Cyt2 toxin can be expressed by itself.…”

Section: Introductionmentioning

confidence: 99%

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

Thammachat¹,

Pungtanom²,

Kidsanguan³

et al. 2010

BMB Reports

Self Cite

View full text Add to dashboard Cite

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]

show abstract

Time influence on the interaction between Cyt2Aa2 and lipid/cholesterol bilayers

Moreno-Cencerrado

Tharad

Iturri

et al. 2016

Microsc. Res. Tech.

Self Cite

View full text Add to dashboard Cite

Protein-membrane interactions are still an important topic of investigation. One of the suitable experimental techniques used by the scientific community to address such question is atomic force microscopy. In a previous work, we have reported that the binding mechanism between the cytolytic and antimicrobial protein (Cyt2Aa2) and lipid/cholesterol bilayers was concentration-dependent, leading to either the formation of holes in the bilayer or aggregates. Here we study such binding mechanism as a function of time at low protein concentrations (10 µg/mL). We demonstrate that although holes are formed during the first stages of the protein-lipid interaction, a reparation process due to molecular mobility in the bilayer leads to a homogenous and isotropic protein-lipid/cholesterol layer within 3 hr. The combination of imaging, force spectroscopy, and phase contrast delivered information about topography dynamics (molecular mobility), layer thickness, and mechanical properties of the protein-lipid/cholesterol system. These results highlight the importance of the observation time in (such type of) protein-lipid interactions (at low protein concentrations).

show abstract

Cloning and Characterization of a Cytolytic and Mosquito Larvicidal δ-Endotoxin from Bacillus thuringiensis subsp. darmstadiensis

Cited by 32 publications

References 0 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

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

Time influence on the interaction between Cyt2Aa2 and lipid/cholesterol bilayers

Contact Info

Product

Resources

About