High-level expression in Escherichia coli, purification and mosquito-larvicidal activity of the binary toxin from Bacillus sphaericus.
The mosquito-larvicidal binary toxin produced by Bacillus sphaericus consists of two polypeptides: BinA and BinB. Both proteins function together, and maximum toxicity is obtained when both are present in equimolar ratio. Cloning and expression of each component separately in heterologous hosts led to low toxicity of the crystal proteins. To improve the expression level, the purification process, and the activity of the binary toxin, the binA and binB genes were separately cloned in Escherichia coli. Each gene was fused in frame to the glutathione S-transferase (GST) gene to be expressed as GST-fusion protein (GST-BinA and GST-BinB). A high expression level was observed from both constructs, and the fusion proteins exhibited high toxicity to Culex quinquefasciatus larvae. High-purity toxin could be obtained by affinity chromatography. The result suggests that GST moiety facilitates high protein production and enables better solubility of the toxin inclusions inside the larval gut, leading to higher toxicity of the fusion protein.
Promdonkoy
Boonhiang
B
National Science and Technology Development Agency, National Center for Genetic Engineering and Biotechnology, 113 Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand. boonhiang@biotec.or.th
Promdonkoy
Peerada
P
Panyim
Sakol
S
D000818:Animals; D001407:Bacillus / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003465:Culex / Q000187:drug effects* / Q000254:growth & development; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007814:Larva / Q000187:drug effects; D007928:Lethal Dose 50; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D016019:Survival Analysis
D001427:Bacterial Toxins; D011993:Recombinant Fusion Proteins; C046134:binB protein, Bacillus sphaericus
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