Two disulfide mutants in domain I of &amp;lt;i&amp;gt;Bacillus thuringiensis&amp;lt;/i&amp;gt; Cry3Aa δ-endotoxin increase stability with no effect on  toxicity

Wu, Sheng Jiun; Florez, Alvaro M.; Homoelle, Bradley J.; Dean, Donald H.; Álzate, Óscar

doi:10.4236/abc.2012.22015

Cited by 1 publication

(1 citation statement)

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“…Variants obtained using rational approximations for Cry11Bb were designed based on the toxin-receptor interaction analyzed in previous studies [7,13,33,35,36]; the Cry11Bb sequence was modified at position 92, changing an alanine for aspartic acid (A92D), and in the second variant, changing the cysteine for arginine at position 157 (C157R). The genes were synthesized at Genescript (Piscataway, NJ, USA) and cloned into the expression vector pSV2, followed by electroporation in E. coli DE3BL21.…”

Section: Cry11bb Mutant Librarymentioning

confidence: 99%

Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis

Suárez-Barrera

Herrera-Pineda

Rondón-Villarreal

et al. 2023

IJMS

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Cry11 proteins are toxic to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of Bacillus thuringiensis and subjected to median-lethal concentration (LC50) tests on first-instar larvae of A. aegypti. LC50 analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL−1), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30–50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against A. aegypti. This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.

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