Efficient solubilization, activation, and purification of recombinant Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli.
A cytotoxic protein Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli was solubilized in 10 mM HCl. Protein concentration of saturated solution of the recombinant Cry45Aa in 10 mM HCl was about 25 times higher than that in the buffer of previous method (in 50 mM sodium carbonate buffer, pH 10.5, containing 1 mM EDTA, and 10 mM dithiothreitol). The Cry45Aa solubilized in the acidic solution was activated by pepsin as an alternative to proteinase K in the previous method. Cytotoxic activity against CACO-2 cells of the pepsin-treated Cry45Aa was almost identical to the proteinase K-treated protein. The pepsin-treated Cry45Aa was purified by cation-exchange chromatography. The concentration of the purified protein was 539 microg/ml, which was 27-fold higher than that of the activated Cry45Aa by the previously method. The cytotoxic activity of the purified protein was stable in broad pH region (pH 2.0-11.0) for 3 days, and 97% cytotoxic activity remained after incubation at 30 degrees C for 360 min.
Okumura
Shiro
S
Fukuoka Industrial Technology Centre, Kurume, Fukuoka 839-0861, Japan. sokumura@fitc.pref.fukuoka.jp
Saitoh
Hiroyuki
H
Wasano
Naoya
N
Katayama
Hideki
H
Higuchi
Kazuhiko
K
Mizuki
Eiichi
E
Inouye
Kuniyo
K
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D018938:Caco-2 Cells; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000633:toxicity; D012995:Solubility
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C412914:parasporin