Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa delta-endotoxin.
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.
Boonserm
Panadda
P
Laboratory of Molecular Biophysics, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Pornwiroon
Walairat
W
Katzenmeier
Gerd
G
Panyim
Sakol
S
Angsuthanasombat
Chanan
C
D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D006460:Hemolysin Proteins; D006868:Hydrolysis; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis