Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa delta-endotoxin.
Autor(es): Boonserm Panadda; Pornwiroon Walairat; Katzenmeier Gerd; Panyim Sakol; Angsuthanasombat Chanan
Resumo: 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 degrees 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. 65kDa. 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.
Palavras-Chave: Bacillus thuringiensis; Toxin-inclusion; Interhelical cleavage; Larvicidal activity; Mutagenesis; Size-exclusion chromatography
Imprenta: Protein Expression and Purification, v. 35, n. 2, p. 397-403, 2004
Identificador do objeto digital: 10.1016/j.pep.2004.02.016
Descritores: Aedes aegypti - Cell ; Aedes aegypti - DNA ; Aedes aegypti - Molecular Structure ; Aedes aegypti - Pathogenesis ; Aedes aegypti - Proteins
Data de publicação: 2004