Publication: Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa δ-endotoxin
Issued Date
2004-01-01
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ISSN
10465928
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2-s2.0-2642583349
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Mahidol University
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SCOPUS
Bibliographic Citation
Protein Expression and Purification. Vol.35, No.2 (2004), 397-403
Suggested Citation
Panadda Boonserm, Walairat Pornwiroon, Gerd Katzenmeier, Sakol Panyim, Chanan Angsuthanasombat Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa δ-endotoxin. Protein Expression and Purification. Vol.35, No.2 (2004), 397-403. doi:10.1016/j.pep.2004.02.016 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/21234
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Title
Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa δ-endotoxin
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Abstract
Achieving high-level expression of the Bacillus thuringiensis Cry4Aa mosquito-larvicidal protein was demonstrated. The 130-kDa Cry4Aa protoxin was overexpressed as an inclusion body in Escherichia coli under the control of the tac promoter together with the cry4Ba promoter. The solubility of the toxin inclusions in carbonate buffer, pH 10.0, was markedly enhanced at a cultivation temperature of 30°C. Elimination of the tryptic cleavage site at Arg-235 in the loop between helices 5 and 6 still retained the high-level toxicity of E. coli cells expressing the Cry4Aa mutant against Aedes aegypti larvae. Trypsin digestion of the R235Q mutant protoxin produced a protease-resistant fragment of ca. 65 kDa. A homogeneous product of the 65-kDa trypsin-treated R203Q protein was obtained after size-exclusion chromatography that would pave the way for the further crystallisation and X-ray crystallographic studies. © 2004 Published by Elsevier Inc.